Compositions and methods for enrichment of neural stem cells using ceramide analogs

ABSTRACT

The present invention provides compositions and methods for human neural cell production. More particularly, the present invention provides cellular differentiation methods employing amphiphilic lipid compounds, preferably ceramide analogs of the β-hydroxyalkylamine type and optionally employing an essentially serum free MEDII conditioned medium for the generation of human neural cells from pluripotent human cells. The methods alternatively comprise modulating apoptosis by modifying the levels of PAR-4, with or without the presence of amphiphilic lipid compounds and optionally employing MEDII conditioned medium. The methods alternatively encompass modulating apoptosis by modulating the intracellular concentration of endogenous lipid second messengers, such as ceramide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase application of PCT/US03/30112,filed Sep. 25, 2003, which claims priority to U.S. Provisional PatentApplication No. 60/413,510 filed on 25 Sep. 2002, and to U.S.Provisional Patent Application No. 60/485,351 filed on Jul. 7, 2003.

ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT

This invention was made, at least in part, with funding from theNational Institutes of Health (Award Number MH064794 to BGC, MH61934-04and 1R01 NS046835-01 to EB). Accordingly, the United States Governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to mammalian stem cells and todifferentiated or partially differentiated cells derived therefrom usingamphiphilic lipid compounds, and preferably, using novel ceramideanalogs of the β-hydroxyalkylamine type. The present invention alsorelates to methods of producing, differentiating and culturing the cellsof the invention, and to uses thereof. The methods alternativelycomprise modulating the levels of apoptotic regulating factors, such asPAR-4, and/or modulating the intracellular concentration of endogenouslipid second messengers, such as ceramide. These further methods canoptionally be performed in the presence of amphiphilic lipid compounds,and optionally employ MEDII conditioned medium. The invention furtherrelates to compositions comprising the amphiphilic lipid analogs andMEDII conditioned medium.

2. Background Art

Embryonic stem (ES) cells represent a powerful model system for theinvestigation of mechanisms underlying pluripotent cell biology anddifferentiation within the early embryo, as well as providingopportunities for genetic manipulation of mammals and resultantcommercial, medical and agricultural applications. Furthermore,appropriate proliferation and differentiation of ES cells can be used togenerate an unlimited source of cells suited to transplantation fortreatment of diseases that result from cell damage or dysfunction. Otherpluripotent cells and cell lines including early primitive ectoderm-like(EPL) cells as described in International Patent Application WO99/53021, in vivo or in vitro derived ICM/epiblast, in vivo or in vitroderived primitive ectoderm, primordial germ cells (EG cells),teratocarcinoma cells (EC cells), and pluripotent cells derived bydedifferentiation, reprogramming or by nuclear transfer will share someor all of these properties and applications.

Human ES cells have been described in International Patent ApplicationWO 96/23362, and in U.S. Pat. Nos. 5,843,780, and 6,200,806; and humanEG cells have been described in International Patent Application WO98/43679, and U.S. Pat. No. 6,245,566.

The ability to tightly control differentiation or form homogeneouspopulations of partially differentiated or terminally differentiatedcells by differentiation in vitro of pluripotent cells has provedproblematic. Most current approaches involve the formation of embryoidbodies from pluripotent cells in a manner that is not controlled anddoes not result in homogeneous populations. Mixed cell populations suchas those in embryoid bodies of this type are generally unlikely to besuitable for therapeutic or commercial use.

Uncontrolled differentiation produces mixtures of pluripotent stem cellsand partially differentiated stem/progenitor cells corresponding tovarious cell lineages. When these ES-derived cell mixtures are graftedinto a recipient tissue the contaminating pluripotent stem cellsproliferate and differentiate to form tumors, while the partiallydifferentiated stem and progenitor cells can further differentiate toform a mixture of inappropriate and undesired cell types. It is wellknown from studies in animal models that tumors originating fromcontaminating pluripotent cells can cause catastrophic tissue damage anddeath. In addition, pluripotent cells contaminating a cell transplantcan generate various inappropriate stem cell, progenitor cell anddifferentiated cell types in the donor without forming a tumor. Thesecontaminating cell types can lead to the formation of inappropriatetissues within a cell transplant. These outcomes cannot be tolerated forclinical applications in humans. Therefore, uncontrolled ES celldifferentiation makes the clinical use of ES-derived cells in human celltherapies impossible.

Selection procedures have been used to obtain cell populations enrichedin neural cells from embryoid bodies. These include genetic modificationof ES cells to allow selection of neural cells by antibiotic resistance(Li et al., 1998 Current Biol., 8:971-974), and manipulation of cultureconditions to select for neural cells (Okabe et al., 1996 Mech., Dev.59:89-102; and Tropepe et al., 2001 Neuron, 30:65-78; O'Shea, 2002 Methin Mol., Biol. 198:3-14). Previously, one research group hasdemonstrated efficient differentiation of mouse and primate ES cells toTH+ neurons following co-culture with the PA6 stromal cell line, butthis technique is not likely to be useful for cell therapy applicationsas it introduces xenograft issues associated with exposure to non-humancell lines and removal of potential PA6 cell contamination in subsequentcultures (Kawasaki et al., 2000 Neuron, 28:31-40; Kawasaki et al., 2002Proc. Natl. Acad. Sci. USA, 99(3):1580-1585). Furthermore, the PA6differentiation procedure generated non-neural terminally differentiatedcell types, such as retinal epithelial cells, reducing the usefulness ofthe cell cultures for cell therapy. In addition, McKay has demonstratedefficient differentiation of mouse ES cells to TH+ neurons, but thisdifferentiation required over-expression of the Nurr-1 transcriptionfactor in combination with exposure to Sonic Hedgehog and FGF8 (Kim etal., 2002 Nature 418(6893):50-6). Furthermore, the McKay protocolinvolves a complex, five stage differentiation method fordifferentiation of mouse ES cells to neurons.

In all of these procedures, the differentiation of pluripotent cells invitro does not involve biological molecules that direct differentiationin a controlled manner. Similarly, in experiments examining neuraldifferentiation from human ES cells, there is no way to control theneural differentiation, and the methods merely allow for the passivedevelopment of neural cell types (see Zhang et al., 2001 Nature Biotech,19(12):1129-1133, and Reubinoff et al., 2001 Nature Biotech,19(12):1134-40). Hence homogeneous, synchronous populations of neuralcells with unrestricted neural differentiation capability are notproduced, restricting the ability to derive essentially homogeneouspopulations of partially differentiated or differentiated neural cells.Another research group differentiated human ES cell derived embryoidbodies in 20% serum containing medium for 4 days followed by plating andselection/expansion of neural cell types in medium containing B27 and N2supplements (serum free), EGF, FGF-2, PDGF-AA, and IGF-1 (Carpenter etal., 2001 Exper. Neuro., 172:383-397). Carpenter et al. showed thatneural progenitors could be enriched from this culture system by cellsorting or immunopanning using antibodies directed against polysialatedNCAM or the cell surface molecule recognized by the A2B5 monoclonalantibody.

Chemical inducers such as retinoic acid have also been used to formneural lineages from a variety of pluripotent cells including ES cells(Bain et al., 1995 Dev. Biol., 168:342-357, Strubing et al., 1995 Mech.Dev., 53:275-287, Fraichard et al., 1995 J. Cell Sci., 108:3181-3188,Schuldhrer et al., 2001 Brain Res., 913:201-205; Esdar et al., 2001 Eur.J. Cell Biol., 80:539-553). However, the route of retinoic acid-inducedneural differentiation has not been well characterized, and therepertoire of neural cell types produced appears to be generallyrestricted to ventral somatic motor, branchiomotor or visceromotorneurons (Renoncourt et al., 1998 Mech. Dev., 79:185-197).

Previous publications report the transplantation of ES-derived neuralcells into the ventricles of the fetal or newborn rat or mouse brainwithout the formation of tumors (Brustle et al., 1997 PNAS,94:14809-14814, Zhang et al., 2001 Nature Biotech, 19:1129-1133).Although some of the cells in these studies do integrate into the hostbrain, many of the cells in the transplants form neural tube likestructures within the lumen of the brain ventricle. Therefore, theseprevious studies do not lead to methods that can be readily applied tohuman cell therapy. Note that Reubinoff et al. (2001 Nature Biotech,19:1134) also injected ES-derived neural cells into the ventricles ofnewborn mice but did not report intraventricular masses of neural cells,omitting any mention of the presence or absence of such masses.

Neural stem cells and precursor cells have been derived from fetal brainand adult primary central nervous system tissue in a number of species,including rodent and human (e.g., see U.S. Pat. No. 5,753,506 (Johe),U.S. Pat. No. 5,766,948 (Gage), U.S. Pat. No. 5,589,376 (Anderson andStemple), U.S. Pat. No. 5,851,832 (Weiss et al.), U.S. Pat. No.5,958,767 (Snyder et al.) and U.S. Pat. No. 5,968,829 (Carpenter).However, each of these disclosures fails to describe a predominantlyhomogeneous population of neural stem cells able to differentiate intoall neural cell types of the central and peripheral nervous systems,and/or essentially homogeneous populations of partially differentiatedor terminally differentiated neural cells derived from neural stem cellsby controlled differentiation. Furthermore, it is not clear whethercells derived from primary fetal or adult tissue can be expandedsufficiently to meet potential cell and gene therapy demands. Neuralstem cells derived from fetal or adult brain are established andexpanded after the cells have committed to the neural lineage and insome cases after the cells have committed to neural sublineages.Therefore, these cells do not provide the opportunity to manipulate theearly differentiation processes that occur prior to neural commitment.Pluripotent stem cells provide access to these earliest stages ofmammalian cellular differentiation opening additional options for cellexpansion and directed development of the cells into desired lineages.

It has been suggested that sphingosine, ceramide and ceramide analogscan be used to induce apoptosis in certain cells; however, the resultsto date have been inconsistent. Ceramide has been reported to induceapoptosis in some cells or cell-lines of neural origin, while in otherreports ceramide application has protected the cells from apoptosis. Forexample, compare Marcora et al., 1996 Found. Clin. Immunol., 4:11-13;Hartfield et al., 1997 FEBS Lett, 401:148-152; Casaccia-Bonnefil et al.,1996 Nature, 383:716-719; Brugg et al., 1996 J. Neurochem., 66:733-739to Furuya et al., 1998 J. Neurochem., 71:366-377; Irie and Hirabayashi,1998 J. Neurosci. Res., 54:475-485; Ito & Horigome, 1995 J. Neruochem.,65:463-466; and Liu et al., 2000 Am. J. Cell Physiol., 278:C144-153. Theresults achieved have been dependent on cell type, cell density, and theconcentration of ceramide or the ceramide analog used (For review, seeToman et al., 2002 J. Neurosci. Res., 68:323-330). These experimentshave been performed on tumor cells, immortalized cell lines, and primarycultures of differentiated cells, and the results have not beenextrapolated to a culture of undifferentiated stem cells. See, Obeid etal., 1993 Science, 259:1769-1771; Marcora et al., 1996 Found. Clin.Immunol., 4:11-13; Hartfield et al., 1997 FEBS Lett, 401:148-152;Casaccia-Bonnefil et al., 1996 Nature, 383:716-719; Herget et al., 2000J. Biol. Chem., 275:30344-30354; and U.S. Pat. No. 6,410,597(Bieberich). It has been postulated that the effects of ceramideapplication may be mediated by a ceramide-activated pathway or feedbackmechanism (Jaffrezou et al., 1998 FASEB J., 12:999-1006.)

The complexity of ceramide-dependent neuronal apoptosis, and theresulting lack of predictability in the prior art is demonstrated by astudy showing the expression profile of 239 genes that respond toC2-ceramide treatment (Decraene et al., 2002 Genome Biol., 3(8):research0042.1-0042.22). This study showed that C2-ceramide treatment bothupregulated and downregulated pro-apoptotic genes in neuronallydifferentiated PC12 cells, indicating a complex and unpredictablegenetic response to C2-ceramide treatment. Further, certain ceramideexperiments have even shown that ceramide induces apoptosis in somedifferentiated neural cells (See Toman et al., 2002 J. Neurosci. Res.,68:323-330; Brugg et al., 1996 J. Neurochem., 66:733-739), suggestingthat the use of ceramide may not be a suitable way to select fordifferentiated or partially differentiated neural cells that candifferentiate into all neural cell types of the central and peripheralnervous systems.

Extensive programmed cell death/apoptosis occurs during neuraldifferentiation in the developing mammalian central nervous system(Chun, J., 2000 Trends in Neuroscience, 23:407-408; Blaschke et al.,1996 Development, 122:1165-1174; Blaschke et al., 1998 J. ComparativeNeurology, 396:39-50). This early cell death appears to be largelyconfined to the neural stem cell and neural progenitor cell pools. Inembryonic day 14 mouse cerebral cortex, 70% of the cells undergoapoptosis/programmed cell death (Blaschke et al., 1996 Development,122:1165-1174). The finding that a large proportion of neural progenitorcells undergo programmed cell death suggests that neural stem/progenitorcells may be extremely responsive to inducers of apoptosis/programmedcell death such as ceramide and ceraride analogs In this context, theresistance of embryonic stem cell-derived neural progenitor/stem cellsto ceramide induced or enhanced cell death would be surprising andunexpected.

In summary, it has not been possible to control the differentiation ofpluripotent cells in vitro, to provide homogeneous, synchronouspopulations of neural cells with unrestricted neural differentiationcapacity. Similarly, methods have not been developed for the derivationof neural cells from pluripotent cells in a manner that parallels theirformation during embryogenesis. In addition, current methods have reliedupon the expression of foreign genes to drive neural differentiation ofpluripotent stem cells (Kim et al., 2002 Nature, 418:50-56). Theselimitations have restricted the ability to form essentially homogeneous,synchronous populations of partially differentiated and terminallydifferentiated neural cells in vitro, and have restricted their furtherdevelopment for therapeutic and commercial applications.

There is a need, therefore, to identify methods and compositions for theproduction of a population of cells enriched in neural stem cells andthe products of their further differentiation, and in particular, humanneural cells and their products.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome, or at leastalleviate, one or more of the difficulties or deficiencies associatedwith the prior art. In that regard, the present invention provides amethod of producing a human neural cell including the steps of: a)providing a pluripotent human cell; b) culturing the pluripotent humancell to form an embryoid body; and c) culturing cells from the embryoidbody with a composition comprising an amphiphilic lipid compound toproduce the human neural cell. The present invention also provides amethod of enriching a human cell culture for neural cells including thefollowing steps: a) providing a pluripotent human cell culture; b)culturing the pluripotent human cell culture to form an embryoid body;and c) culturing cells from the embryoid body with a compositioncomprising an amphiphilic lipid compound to produce a human cell cultureenriched in neural cells. The invention further contemplates that theamphiphilic lipid compound is selected from the group consisting of aceramide compound, a sphingosine compound, and a hydroxyalkyl estercompound. In a preferred embodiment, the pluripotent human cell orpluripotent human cell culture is a differentiating cell or cellculture. In certain preferred embodiments, the pluripotent human cell orcell culture is cultured with an essentially serum free medium to forman embryoid body. In a preferred embodiment, the essentially serum freemedium is a MEDII conditioned medium, and/or contains proline, or aproline containing peptide.

The invention further encompasses methods for modulating apoptosis inhuman pluripotent cell populations comprising modifying expression of anapoptotic regulating factor, and/or modulating the intracellularconcentration of endogenous lipid second messengers, such as ceramide.Preferably, the apoptotic regulating factor is PAR-4. Preferably,modulating expression of PAR-4 comprises increasing or decreasing themRNA levels and/or the protein levels of PAR-4. Increasing expression ofPAR-4 mRNA and/or protein is correlated with apoptosis, while decreasingexpression of PAR-4 mRNA and/or protein is correlated with a decrease inapoptosis. PAR-4 expression can be modulated in pluripotent cellpopulations, or can be modulated in differentiated orpartially-differentiated cell populations. Modulation of the expressionof PAR-4 is optionally performed in the presence of an amphiphilic lipidcompound or ceramide, and optionally can employ MEDII conditionedmedium.

The amphiphilic lipid compound described herein can be preferably aceramide analog of the hydroxyalkylamine type. In preferred embodiments,the ceramide compound is selected from the group comprisingN-(2-hydroxy-1-(hydroxymethyl)ethyl)-palmitoylamide (“S16”);N-(2-hydroxy-1-(hydroxymethyl)ethyl)-oleoylamide (“S18”); and functionalhomologues, isomers, and pharmaceutically acceptable salts thereof. In apreferred embodiment the ceramide compound is S18. In another preferredembodiment the ceramide compound is S16.

The MEDII conditioned medium described herein can be preferably a Hep G2conditioned medium that contains a bioactive component selected from thegroup consisting of a low molecular weight component; a biologicallyactive fragment of any of the aforementioned proteins or components; andan analog of any of the aforementioned proteins or components. In apreferred embodiment, the bioactive component of the MEDII conditionedmedium is proline, or a proline containing peptide. The pluripotenthuman cell of the present invention can be selected from, but is notlimited to, a human embryonic stem cell; a human ICM/epiblast cell; anEPL cell; a human primitive ectoderm cell; a human primordial germ cell;and a human EG cell.

In other embodiments of the present invention, the methods describedabove further include the steps of dispersing the embryoid body to anessentially single cell suspension, and culturing the essentially singlecell suspension comprising the pluripotent human cell with a compositioncomprising a ceramide compound until the human neural cell is produced,or the human cell culture enriched in neural cells is produced.

The invention further provides a composition comprising a culture ofneural cells derived in vitro from a pluripotent human cell culturedwith a composition comprising a ceramide compound. In preferredembodiments, these neural cells are capable of expressing one or more ofthe detectable markers for tyrosine hydroxylase (TH), vesicular monaminetransporter (VMAT) dopamine transporter (DAT), and aromatic amino aciddecarboxylase (AADC).

The invention further provides a method of treating a patient with aneural disease, comprising a step of administering to the patient atherapeutically effective amount of the neural cell or cell cultureenriched in neural cells produced using the methods of the presentinvention. The present invention further provides a method for enhancingthe efficiency of the transplantation of a cultured human pluripotentcell. In a preferred embodiment, the method comprises culturing a humanpluripotent cell with a growth medium comprising an amphiphilic lipidcompound, and transplanting the cultured human pluripotent cell into apatient. The amphiphilic lipid compound preferably can be selected fromthe group consisting of a ceramide compound, a sphingosine compound, anda hydroxyalkyl ester compound. In one embodiment, the ceramide compoundis a ceramide analog of the hydroxyalkylamine type. In a preferredembodiment, the ceramide compound is selected from the group comprisingS16, S18 and functional homologues, isomers, and pharmaceuticallyacceptable salts thereof.

The invention further provides compositions for promoting themaintenance, proliferation, or differentiation of a human neural cell,the composition comprising a cell culture medium comprising MEDIIconditioned medium or a bioactive component thereof, and a ceramidecompound. In a preferred embodiment, the ceramide compound is selectedfrom the group comprising S16, S18 and functional homologues, isomers,and pharmaceutically acceptable salts thereof. The invention alsoprovides for compositions comprising a cell culture medium comprisingMEDII conditioned medium and an amphiphilic lipid compound selected fromthe group consisting of a sphingosine compound, and a hydroxyalkyl estercompound. In one preferred embodiment, the hydroxyalkyl ester compoundis laurylgallate. The invention further provides for the neural cellscultured in the compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-G show the chemical structure of ceramide, and novel structuralanalogs of ceramide (novel ceramide analogs or NCAs) synthesized byN-acylation of β-hydroxyalkylamines. A shows the chemical structure ofN-acyl sphingosine (“ceramide”). B shows the chemical structure ofN-(2-hydroxy-1-hydroxymethyl)ethyl)-palmitoylamide (“S16”). C shows thechemical structure of N-(2-hydroxy-1-(hydroxymethyl)ethyl)-oleoylamide(“S18”). D shows the chemical structure ofN,N-bis(2-hydroxyethyl)palmitoylamide (“B16”). E shows the chemicalstructure of N,N-bis(2-hydroxyethyl)oleoylamide (“B18”). F shows thechemical structure of N-tris(hydroxymethyl)methyl-palmitoylamide(“T16”). G shows the chemical structure ofN-tris(hydroxymethyl)methyl-oleoylamide (“T18”).

FIG. 2 is a schematic showing the in vitro neural differentiation ofmouse embryonic stem cells. Abbreviations: ES (embryonic stem cell); EB(embryoid body); NP (neural progenitor cell); D (terminallydifferentiated cell); NEP (neuroepithelial precursor cell); GRP (glialrestricted precursor cell); NRP (neuronal restricted precursor cell);LIF (leukemia inhibitory factor); DIV (days in vitro); FGF-2 (fibroblastgrowth factor 2); N2 (medium supplement N2); and Oct-4, GFAP, and MAP-2,are markers for differentiation proteins.

FIG. 3 shows the levels of spontaneous and induced apoptosis indifferentiating ES-J1 cells. During particular stages of in vitro neuraldifferentiation, apoptosis was induced in ES-J1 cells by incubation for20 hours with 35 μM C2-ceramide, 75 μM S18, or 100 μM S16. Apoptosis wasdetermined by TUNEL staining. The levels of apoptosis in ceramidetreated samples was compared to the levels in control samples that werenot incubated with ceramide analogs. Each experiment was performed fivetimes. The bars show the standard mean and deviation of % TUNEL positivecells that were counted in five areas of 200 cells in each experiment.Open bars, no ceramide analog treatment; black bars, ceramide analogtreatment.

FIGS. 4A-J show the cell death of ES-J1 cells treated with the novelceramide analog S18 during in vitro neural differentiation. FIGS. 4A andB show cell death in ES cells without and with S18 incubation,respectively. FIGS. 4C and D show cell death at the EB4 stage withoutand with S18 incubation, respectively. FIGS. 4E and F show cell death atthe EB8 stage without and with S18 incubation, respectively. FIGS. 4Gand H show cell death at the NP2 stage without and with S18 incubation,respectively. FIGS. 4I and J show cell death in differentiated neuronswithout and with S18 incubation, respectively. ES-J1 cells weredifferentiated in vitro following the protocol as described herein, andwere subsequently incubated for 20 hours with 75 μM of the novelceramide analog S18. Note the high degree of cell death that was inducedat the EB8 (E, and F) and NP2 stages (G, and H), whereas differentiatedneurons were unaffected by ceramide treatment (compare I to J). Notealso that at the EB8 stage, a rim of cells surrounding the centralembryoid body survived treatment with ceramide analogs. See FIG. 2 foran explanation of the differentiation stages.

FIGS. 5A, and B show Hoechst staining and nestin antibody staining ofmouse EB8 cells after incubation with S18. Differentiating embryonicstem cells at stage EB8 were incubated for 24 hours with 80 μM of S18,and were then immunostained for nestin. Apoptosis was detected byintensive staining with Hoechst dye. Note that the center of theembryoid body (left side of A) stained strongly with Hoechst 33258 andindicates apoptotic cells, whereas the rim of non-apoptotic cells in theembryoid body stained intensively for nestin (13).

FIG. 6 shows a table summarizing double staining results for TUNEL andvarious marker proteins at the NP2 stage. TUNEL staining detectsapoptotic cells, and the marker proteins indicate the stage of neuraldifferentiation. The total number of cells staining for one specificantigen within a population of 200 cells was as follows: TUNEL, 65;PAR-4, 91; ceramide, 105; nestin, 113; and PCNA, 108. The table showsthe number of cells that stained simultaneously for two antigens. Notethat the TUNEL positive cells co-localized significantly less withnestin (8% of TUNEL positive cells were nestin positive cells while 57%of the total cell population was nestin positive cells) and that theTUNEL positive cells co-localized significantly more with PCNA (74% ofTUNEL positive cells were PCNA positive cells while 54% of the totalcell population was PCNA positive cells). A chi square analysis of thesedistributions showed that TUNEL positive cells were predominantly nestinnegative and PCNA positive. The abbreviation “n.d.” indicates that aparticular combination was not determined.

FIGS. 7A and B show that EB-derived stem cells treated with novelceramide analogs of the serinol type do not form teratomas when injectedinto neonate mouse brains. Ten days after injection of the untreated EScells (A) or treated ES cells (B), the brains were isolated foranalysis. Massive teratoma formation was observed with untreated,control cells (A), while EB8-derived cells that have been treated withS18 did not show the formation of teratomas (3). The black India inkspot on the right side of the brain in panel B marks the injectionchannel.

FIGS. 8A-H show teratoma formation with untreated ES cells and tissueintegration with S18-treated ES cells. EB8-derived stem cells werestained with a fluorescent marker dye (Vybrant diI) in order to trackthe migration and integration of the injected cells into the recipient'sbrain tissue. A and B show the injection site of untreated EB8-derivedembryonic stem cells, while E and F show the injection site of S18treated EB8-derived embryonic stem cells. C and D show the migrationsite of untreated EB8-derived embryonic stem cells, while G and H showthe migration site of S18 treated EB8-derived embryonic stem cells.Brains injected with untreated cells show teratoma formation anddisplacement growth at the migration site (C and D). Only the center ofthe tumor is stained with Vybrant diI. In the periphery of the tumor,cells have undergone numerous cell divisions, resulting in dilution ofthe fluorescent dye and low levels of staining. Note the bright VybrantdiI staining of cells that have integrated into the recipient's braintissue (G and H). This intensive staining indicates that the cells haveundergone a limited number of cell divisions.

FIGS. 9A-D show expression of Oct-4 protein in HESCs and serum beembryoid bodies. A shows high levels of Oct-4 expression in a typicalmanually passaged HESC colony, with distinct nuclear expression inundifferentiated ES cells and no Oct-4 in the unstained feeder layersurrounding the HESC colony. B shows a typical manually passaged HESCcrater colony, showing high levels of Oct-4 expression in themultilayered ring of undifferentiated cells surrounding the monolayercrater cells that express a low level of Oct-4. Differentiating cells atthe edge of the colony also express a low level of Oct-4. C shows theexpression of Oct-4 in a seeded essentially serum free embryoid body,representative of what is seen when sfEBMs are derived from domed HESCsor monolayer crater cells. Regions of high level Oct-4 expressionpersist and are indicative of residual nests of pluripotent cellsmaintained by local cell-cell signaling events. Neural rosettes in thesame field are indicated as radially organized circles of nuclei by DAPIstaining (D) and these neural precursor cells only express low levels ofOct-4.

FIGS. 10A-E show the effect of S18 treatment on seeded sfEBMs. A shows aseeded essentially serum free embryoid body exhibiting neural rosetteswithin the core of the explant and other cell types that haveproliferated away from the rosettes. B shows that a high proportion ofcells within these cultures have been killed after 36 hours exposure to6 μM S18. C shows that a high degree of cell death is apparent after 36hours exposure to 8 μM S18. Neural rosettes appear to be unaffected andin many cases can be observed more clearly, as surrounding cell typeshave died. D is a 60× magnification of surviving neural rosette after 36hours exposure to 8 μM S18. The rosette appears morphologically normaland the typical radial organization of cells and distinct boundarybetween healthy rosette cells and apoptotic surrounding cells can beobserved. E shows that the dying cells are undergoing apoptosis.Apoptosis of dying cells is indicated by their fragmented nuclei whenstained with DAPI. Morphologically normal nuclei of unaffected cells arepresent in the lower right corner.

FIGS. 11A and B demonstrate the purification of neural rosette materialby exposure of sfEBMs in suspension to S18. A shows S18 resistant neuralrosette material isolated from generally degenerating sfEBMs grown insuspension at 20×. B shows a 40× magnification of a different piece ofS18 resistant neural rosette material.

FIGS. 12A and B show the ablation of residual pluripotent cells in sfEBMcultures exposed to S18. sfEBM cultures exposed to S18 in suspension,followed by seeding and immunocytochemistry do not exhibit any cellsexpressing high levels of Oct-4. This demonstrated that residual nestsof pluripotent cells did not survive S18 induced apoptosis.

FIGS. 13A-F show that neural rosette cells are unaffected by exposure toS18. FIGS. 13A, B, and C show the same field of seeded sfEBMs stainedwith anti-Oct-4, anti-Map2 and anti-TH, respectively. Seeded rosettecells only express low levels of Oct-4 (A) and mature neurons (Map2+; B)are either also resistant to S18 or are regenerated effectively from therosette precursor cells. A proportion of the Map2+ cells arepresumptively dopaminergic neurons as they express Tyrosine Hydroxylase(C), indicating that they are also resistant to S18 and/or the rosetteprecursor cells maintain their capacity to differentiate to dopaminergicneurons. D and E show 40× magnification of Map2 and TH positive neuronsin the same field, respectively. F shows that neural rosettes were stillproliferative after exposure to S18, as demonstrated by phosphoHistoneH3 staining for mitotic cells (indicated as the intense white spots)within DAPI stained rosettes, shown as the paler staining radiallyorganized structures.

FIGS. 14A-D show sections of sfEBMs exposed to S18. The sfEBMs werederived from protease passaged HESCs exposed to 10 μM S18 from day 6 to9 after derivation of the embryoid bodies. Sections were stained withDAPI to reveal rosette organization and nuclear morphology. A shows asection of an untreated sfEBM at day 9, while B-D show sections ofsfEBMs at day 9 that were treated with S18 from days 6-9.

FIG. 15 shows in vitro neuronal differentiation of embryonic stem cells,overview and marker protein expression. Immunostaining for markerproteins was performed with protein extracts from stem cells at thedifferentiation stages shown in FIG. 2.

FIGS. 16A and 16B show ceramide content and apoptosis during in vitroneuronal differentiation of ES cells. In 16A, neutral lipids werepurified from differentiating ES cells and a lipid amount correspondingto 750 μg of cellular protein/lane separated by HPTLC in the runningsolvent CH₃Cl/HOAc (9:1, by volume). Lipids were stained with the cupricacetate reagent. Ceramide (open bars) was quantified by densitometricanalysis and comparison with known amounts of standard lipid (N-oleoylsphingosine). Lane 1, ceramide standard from bovine brain; lane 2,fibroblast-freed ES-cells, after four days in culture; lane 3, embryoidbodies at the EB4 stage; lane 4, embryoid bodies at the EB8 stage; lane5, neural progenitors at the NP2 stage; lanes 6-8, three terminaldifferentiation stages, 24 h (D1 stage), 48 h (D2 stage), and 96 h (D4stage) upon cultivation of NPs in serum containing medium; lanes 9-11,N-oleoyl sphingosine, 250 ng, 500 ng, 1000 ng. In 16B, lipids wereextracted from differentiated ES cells and the amount of ceramidequantified using the DAG kinase assay. Apoptosis (solid bars) wasdetermined by TUNEL staining. For HPTLC, DAG kinase, and TUNEL analyses,experiments were performed with five independent ES cultures. The barsshow the standard mean and deviation of % TUNEL positive cells that werecounted in five areas of 200 cells in each experiment.

FIG. 17 shows alteration of neural stem cell apoptosis by antisenseknockdown or overexpression of PAR-4. The figure shows staining of PAR-4on immunoblots of protein from differentiating ES cells (NP3 stage) thatwere transfected with or without PAR-4 specific antisenseoligonucleotide or PAR-4-RFP, respectively. Lane 1, untransfected NPs;lane 2, NPs transfected with standard control antisense oligonucleotide;lane 3, NPs transfected with PAR-4 specific antisense oligonucleotide;lane 4, NPs cells transfected with PAR-4-RFP.

FIGS. 18A and 18B show expression of differentiation markers and pro- oranti-apoptotic proteins in differentiating embryonic stem cells. In FIG.18 A, during in vitro neural differentiation of ES-J1 mouse embryonicstem cells, protein was extracted from the cells, separated by SDS-PAGE,and blotted onto nitrocellulose. Each lane shows the immunostainingcorresponding to 35 μg of cell protein. See FIG. 2 for definition ofdifferentiation stages. The protein analysis was performed with fiveindependent differentiation experiments. In FIG. 18B, RNA was isolatedfrom differentiating ES-J1 cells and subjected to RT-PCR. SPT1/2, serinepalmitoyltransferase subunit 1 and 2. Each experiment was repeated fourtines.

FIG. 19 shows correlation of TUNEL and expression of protein markers inindividual cells at the NP2 stage. Cells at the NP2 stage were grown oncoverslips and fixed. TUNEL staining (green fluorescence) was followedby indirect immunofluroescence staining using secondary antibodies thatwere linked to Cy3/Alexa 546 (red fluorescence) or Cy5 (far red). Forspecies of primary antibodies see Materials and Methods. Hoechststaining was used for the identification of individual cells. FIG. 19shows the number of cells (total cell count of 200 in five independentareas) that were TUNEL positive (average of 63+/−4) or negative (averageof 137+/−11) and stained for another marker as indicated (percentage ofTUNEL positive or negative is given in brackets). FIG. 19 shows standardmeans and variation for five areas with 200 cells from three independentexperiments.

FIG. 20 shows double-staining experiments for two markers as indicated(A+B represents cells that are double-stained for A and B). Thepercentage of cells in the last column has been calculated from thefrequency of each marker (A or B) in the total population of cells asdescribed in the section Statistical Analysis. FIG. 20 shows standardmeans and variation for five areas with 200 cells from three independentexperiments.

FIG. 21 shows a model for asymmetric apoptosis of neural progenitordaughter cells due to the asymmetric distribution of nestin and PAR-4proteins. Prior to mitosis or during S-phase, neural progenitor cellsup-regulate the expression of nestin, PAR-4, and ceramide. During celldivision, ceramide is distributed equally to the daughter cells, whereasPAR-4 and nestin are restricted to different daughter cells. Thedaughter cell with simultaneous presence of PAR-4 and ceramide will diedue to apoptosis, whereas the one containing ceramide and nestin willagain divide or differentiate. Conversion of ceramide to sphingomyelinand/or glycoshingolipids due to upregulation of sphingomyelin orglucosyl- or galactosylceramide biosynthesis protects this cell fromapoptosis upon further cell division or differentiation.

DETAILED DESCRIPTION OF THE INVENTION

Applicant has demonstrated that culturing human cell populationscomprising pluripotent human cells with a composition comprising anamphiphilic lipid compound results in the formation of a human neuralcell type with greater homogeneity than observed in a pluripotent humancell population that is not cultured with an amphiphilic lipid compound.The invention provides that, alternatively, the human cell populationcultured with the amphiphilic lipid compound is enriched in human neuralcells. In preferred embodiments, the human cell population cultured withthe amphiphilic lipid compound is a differentiating human cellpopulation. In preferred embodiments, the amphiphilic lipid compound isselected from the group comprising a ceramide compound, a sphingosinecompound, and a hydroxyalkyl ester compound. In a preferred embodiment,the composition comprises a ceramide compound of the β-hydroxyalkylaminetype. The invention further provides a partially differentiated cell byculturing pluripotent cells with the ceramide compound.

The invention further encompasses methods for modulating apoptosis inhuman pluripotent cell populations comprising modifying expression of anapoptotic regulating factor, and/or modulating the intracellularconcentration of endogenous lipid second messengers, such as ceramide.Preferably, the apoptotic regulating factor is PAR-4. Preferably,modulating expression of PAR-4 comprises increasing or decreasing themRNA levels and/or the protein levels of PAR-4. Increasing expression ofPAR-4 mRNA and/or protein is correlated with apoptosis, while decreasingexpression of PAR-4 mRNA and/or protein is correlated with a decrease inapoptosis. PAR-4 expression can be modulated in pluripotent cellpopulations, or can be modulated in differentiated orpartially-differentiated cell populations. These methods can optionallybe combined with the other methods and compositions described herein.Modulating the expression of PAR-4 is optionally in the presence of anamphiphilic lipid compound or ceramide, and optionally the methodemploys MEDII conditioned medium. Alternatively, apoptosis is modulatedthrough increasing or decreasing the levels of endogenous lipid secondmessengers, such as ceramide. Levels of endogenous second messengers canbe modulated using compounds and methods well known to those of skill inthe art, including, but not limited to treatment with retinoic acid, theuse of anti-cancer drugs such as daunomycin, and serum deprivation.

In one embodiment, the neural cell produced by culturing the pluripotenthuman cell with a ceramide compound is therapeutically transplanted intothe brain of a subject. The cell culture of the present invention formteratomas at a greatly reduced frequency than if the culture was nottreated with the ceramide compound. In a preferred embodiment, the cellculture of the present invention does not induce the formation ofteratomas at a significant rate.

The present invention particularly provides a method of producing ahuman neural cell that includes the steps of: a) providing a pluripotenthuman cell; and b) culturing the pluripotent human cell with acomposition comprising a ceramide compound to produce a human neuralcell. The present invention additionally provides a method of enrichinga human cell culture for neural cells comprising the steps of a)providing a pluripotent human cell culture; and b) culturing thepluripotent human cell culture with a composition comprising a ceramidecompound to produce a human cell culture enriched in neural cells. In apreferred embodiment, the pluripotent human cell or cell culture is adifferentiating pluripotent human cell or cell culture.

Unless otherwise noted, the terms used herein are to be understoodaccording to conventional usage by those of ordinary skill in therelevant art. In addition to the definitions of terms provided below,definitions of common terms in molecular biology may also be found inRieger et al., 1991 Glossary of genetics: classical and molecular, 5thed, Berlin: Springer-Verlag; in Current Protocols in Molecular Biology,F. M. Ausubel et al., eds., Current Protocols, a joint venture betweenGreene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1998Supplement); in Current Protocols in Cell Biology, J. S. Bonifacino etal., eds., Current Protocols, John Wiley & Sons, Inc. (1999 Supplement);and in Current Protocols in Neuroscience, J. Crawley et al., eds.,Current Protocols, John Wiley & Sons, Inc. (1999 Supplement). It is tobe understood that as used in the specification and in the claims, “a”or “an” can mean one or more, depending upon the context in which it isused. Thus, for example, reference to “a cell” can mean that at leastone cell can be utilized.

The present invention contemplates the use of a composition comprisingan amphiphilic lipid compound. In a preferred embodiment, theamphiphilic lipid compound is selected from the group consisting of aceramide compound, a sphingosine compound, and a hydroxyalkyl estercompound.

In a preferred embodiment, the amphiphilic lipid compound is a ceramidecompound, wherein the ceramide compound is a N-acyl derivative ofβ-hydroxyalkylamine. In a preferred embodiment, the ceramide compoundhas the general formula

and, wherein R is a saturated or mono- or polyunsaturated (cis or trans)alkyl group having greater than 2 carbon atoms; R1, R2, R3, and R4 maybe the same or different and are saturated or mono-or polyunsaturatedhydroxylated alkyl groups, aryl groups, or hydrogen. In one embodiment,R4 is an alkyl chain having from 1 to 12 carbon atoms. In a preferredembodiment, R is a saturated or mono- or polyunsaturated (cis or trans)alkyl group having from 12-20 carbon atoms, the hydroxylated alkylgroups have from 1-6 carbon atoms, R1 and R2 are hydroxylated alkylgroups, and R3 is hydrogen.

In another embodiment, the present invention contemplates the use of acomposition comprising a sphingosine compound, wherein the sphingosinecompound has the general formula

and, R is a saturated or mono- or polyunsaturated (cis or trans) alkylgroup having greater than 2 carbon atoms; R1, R2, R3, and R4 may be thesame or different and are saturated or mono-or polyunsaturatedhydroxylated alkyl groups, aryl groups, or hydrogen. In preferredembodiments, the sphingosine compound is selected from the groupcomprising D-erythro-sphingosine, L-threo-sphingosine,dimethylsphingosine, and N-oleoyl ethanolamine.

In another embodiment, the present invention contemplates the use of acomposition comprising a hydroxyalkyl ester compound, wherein thehydroxyalkyl ester compound has the general formula

and, wherein R is a saturated or mono- or polyunsaturated (cis or trans)alkyl group having greater than 2 carbon atoms; and R1 is a saturated ormono-or polyunsaturated hydroxylated alkyl group, aryl group, orhydrogen. In a preferred embodiment, the hydroxyalkyl ester compound isan O-acyl derivative of gallic acid. In another preferred embodiment,the hydroxyalkyl ester compound is the n-dodecyl ester of3,4,5-trihydroxybenzoic acid (“laurylgallate”), which has the formula

In preferred embodiments of the present invention, the compositioncomprises a ceramide compound selected from the group consisting ofN2-hydroxy-1-(hydroxymethyl)ethyl)-palmitoylamide (“S16”);N-(2-hydroxy-1-(hydroxymethylethyl)-oleoylamide (“S18”);N,N-bis(2-hydroxyethyl)palmitoylamide (“B16”);N,N-bis(2-hydroxyethyl)oleoylamide (“B18”);N-tris(hydroxymethyl)methyl-palmitoylamide (“T16”);N-tris(hydroxymethyl)methyl-oleoylamide (“T18”); N-acetyl sphingosine(“C2-ceramide”);D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol(“D-threo-PDMP”);D-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol(“D-Threo-PPMP”); D-erythro-2-tetradecanoyl-1-phenyl-1-propanol(“D-MAPP”); D-erythro-2(N-myristoylamino)-1-phenyl-1-propanol (“MAPP”),and N-hexanoylsphingosine (C6-ceramide).

Those of skill in the art will recognize that many other variations ofthe general formulas above exist, and that the use of all suchvariations is encompassed by the methods of the present invention. Inmore preferred embodiments, the ceramide compound is selected from thegroup comprising S16, S18 and functional homologies, isomers, andpharmaceutically acceptable salts thereof. In a preferred embodiment theceramide compound is S18. In another preferred embodiment the ceramidecompound is S16. In another preferred embodiment, the amphiphilic lipidcompound can include the metabolites and catabolites of the ceramidecompound, the sphingosine compound, and the hydroxyalkyl ester compound.The composition comprising the amphiphilic lipid compound may furthercomprise pharmaceutically acceptable carriers, excipients, additives,preservatives, and buffers.

In the methods of the present invention, it is preferred that theconcentration of the amphiphilic lipid compound is from approximately0.1 μM to 1000 μM, more preferred that the concentration of theamphiphilic lipid compound is from approximately 1 μM to 100 μM, morepreferred that the concentration of the amphiphilic lipid compound isfrom approximately 5 μM to 50 μM, and most preferred that theconcentration of the amphiphilic lipid compound is approximately 10 μM.

In the methods of the present invention, it is preferred that theduration of culturing the differentiating human pluripotent cell withthe amphiphilic lipid compound is from approximately 1 hour to 20 days,more preferably from approximately 6 hours to 10 days, and mostpreferably from approximately 12 hours to 6 days.

In a preferred embodiment, the pluripotent cell is a human cell. As usedherein, the term “pluripotent human cell” encompasses pluripotent cellsobtained from human embryos, fetuses or adult tissues. In a preferredembodiment, the pluripotent human cell is a differentiating cell. In onepreferred embodiment, the pluripotent human cell is a human pluripotentembryonic stem cell. In preferred embodiments, the human pluripotentembryonic stem cell is obtained from a domed human embryonic stem cellcolony, a crater human embryonic stem cell colony, and a proteasepassaged human embryonic stem cell colony. In another embodiment thepluripotent human cell is a human pluripotent fetal stem cell, such as aprimordial germ cell. In another embodiment the pluripotent human cellis a human pluripotent adult stem cell. As used herein, the term“pluripotent” refers to a cell capable of at least developing into oneof ectodermal, endodermal and mesodermal cells. In one preferredembodiment, the pluripotent human cell is a differentiating human cell.As used herein the term “pluripotent” refers to cells that aretotipotent and multipotent. As used herein, the term “totipotent cell”refers to a cell capable of developing into all lineages of cells. Asused herein, the term “multipotent” refers to a cell that is notterminally differentiated. In one preferred embodiment the multipotentcell is a neural precursor cell and the multipotent cell culture is aneural precursor cell culture. The pluripotent human cell can beselected from the group consisting of a human embryonic stem (ES) cell;a human inner cell mass (ICM)/epiblast cell; a human primitive ectodermcell, such as an early primitive ectoderm cell (EPL); and a humanprimordial germ (EG) cell. The human pluripotent cells of the presentinvention can be derived using any method known to those of skill in theart at the present time or later discovered. For example, the humanpluripotent cells can be produced using de-differentiation and nucleartransfer methods. Additionally, the human ICM/epiblast cell or theprimitive ectoderm cell used in the present invention can be derived invivo or in vitro. EPL cells may be generated in adherent culture or ascell aggregates in suspension culture, as described in WO 99/53021,herein incorporated by reference in its entirety.

In preferred embodiments of the above methods, the method the providesfor an intermediate step of forming an embryoid body comprising thepluripotent human cell. In one embodiment, the embryoid body is formedby culturing the pluripotent human cell or cell culture with anessentially serum free medium. In a preferred embodiment, theessentially serum free medium is a MEDII conditioned medium as definedherein. In other preferred embodiments, the embryoid body issubsequently cultured with one or more cell differentiation environmentsto produce a human neural cell or human cell culture enriched in neuralcells, wherein each environment is appropriate to the cell types as theyappear from the preceding cell type. It is to be understood that theabsence of the term “differentiation” when describing a MEDIIconditioned medium does not indicate that the MEDII conditioned mediumcan not also be considered a “differentiation” environment. In certainembodiments, the essentially serum free medium preferably is alsoessentially LIF free. In a preferred embodiment, a subsequent celldifferentiation environment comprises an amphiphilic lipid compound. Ina preferred embodiment, the amphiphilic compound is selected from thegroup comprising a ceramide compound, a sphingosine compound, and anhydroxyalkyl ester. In more preferred embodiments, the ceramide compoundis a ceramide analog of the serinol type selected from the groupcomprising S16, S18 and functional homologues, isomers, andpharmaceutically acceptable salts thereof. In a preferred embodiment theceramide compound is S18. In another preferred embodiment the ceramidecompound is S16. In a preferred embodiment, the composition comprisingthe amphiphilic lipid compound is essentially serum free.

As used herein, the term “MEDII conditioned medium” refers to a mediumcomprising one or more bioactive components as described herein. In apreferred embodiment, the bioactive component is derived from a hepaticor hepatoma cell or cell line culture supernatant. The hepatic orhepatoma cell or cell line can be from any species, however, preferredcell lines are mammalian or avian in origin. The hepatic or hepatomacell line can be selected from, but is not limited to, the groupconsisting of: a human hepatocellular carcinoma cell line such as a HepG2 cell line (ATCC HB-8065) or Hepa-1c1c-7 cells (ATCC CRL-2026); aprimary embryonic mouse liver sell line; a primary adult mouse livercell line; a primary chicken liver cell line; and an extraembryonicendodermal cell line such as END-2 and PYS-2. A particularly preferredcell line is the Hep G2 cell line (ATCC HB-8065). A description of theisolation of an essentially serum free MEDII conditioned medium from aHep G2 cell line is provided in Example 2 below. In one embodiment ofthe present invention, the MEDII conditioned medium is derived from aHep G2 cell line and contains supplements of FGF-2.

As used herein, “essentially serum free” refers to a medium that doesnot contain serum or serum replacement, or that contains essentially noserum or serum replacement. As used herein, “essentially” means that ade minimus or reduced amount of a component, such as serum, may bepresent that does not eliminate the improved bioactive neural cellculturing capacity of the medium or environment. For example,essentially serum free medium or environment can contain less than 10,9, 8, 7, 6, 5, 4, 3, 2, or 1% serum wherein the presently improvedbioactive neural cell culturing capacity of the medium or environment isstill observed.

As used herein, “essentially LIF free” refers to a medium that does notcontain leukemia inhibitory factor (LIF), or that contains essentiallyno LIF. As used herein, “essentially” means that a de mininius orreduced amount of a component, such as LIF, may be present that does noteliminate the improved bioactive neural cell culturing capacity of themedium or environment. For example, essentially LIF free medium orenvironment can contain less than 100, 75, 50, 40, 30, 10, 5, 4, 3, 2,or 1 ng/ml LIF, wherein the presently improved bioactive neural cellculturing capacity of the medium or environment is still observed.

As used herein, the terms “bioactive component” and “bioactive factor”refer to any compound or molecule that induces a pluripotent cell tofollow a differentiation pathway toward an EPL cell or a neural cell.Alternatively, the bioactive component may act as a mitogen or as astabilizing or survival factor for a cell differentiating towards an EPLcell or neural cell. A bioactive component from the conditioned mediummay be used in place of the MEDII conditioned medium in any embodimentdescribed herein. The isolation of a bioactive component of MEDII isshown below in Example 2. While the bioactive component may be asdescribed below, the term is not limited thereto. The term “bioactivecomponent” as used herein includes within its scope a natural orsynthetic molecule or molecules which exhibit(s) similar biologicalactivity, e.g. a molecule or molecules which compete with moleculeswithin the conditioned medium that bind to a receptor on ES or EPL cellsor their differentiation products in adherent culture, in embryoidbodies, or in nonadherent cultures, responsible for EPL or neuralinduction, and/or EPL or neural proliferation, and/or EPL or neuralsurvival.

The MEDII conditioned medium described herein can comprise one or morebioactive components selected from the group consisting of a lowmolecular weight component comprising proline or a proline containingpeptide; a biologically active fragment of any of the aforementionedproteins or components; and an analog of any of the aforementionedproteins or components. In addition, the MEDII conditioned medium maycontain a neural inducing factor.

The low molecular weight component of the MEDII conditioned medium cancomprise one or more proline residues or a polypeptide containingproline residues. As used herein, the term “polypeptide” refers to anyof various amides that are derived from two or more amino acids bycombination of the amino group of one acid with the carboxyl group ofanother and usually obtained by partial hydrolysis of proteins. In apreferred embodiment, the low molecular weight component is L-proline ora polypeptide including L-proline. The proline containing polypeptidepreferably has a molecular weight of less than approximately 5 kD, morepreferably less than approximately 3 kD. In a further preferredembodiment, the low molecular weight component is a polypeptide ofbetween approximately 2-11 amino acids, more preferably of betweenapproximately 2-7 amino acids and most preferably approximately 4 aminoacids. The proline containing polypeptide can be selected from, but isnot limited to, the following polypeptides: Pro-Ala, Ala-Pro,Ala-Pro-Gly, Pro-OH-Pro, Pro-Gly, Gly-Pro, Gly-Pro-Ala, Gly-Pro-Glu,Gly-Pro-OH-Pro, Gly-Pro-Arg-Pro (SEQ ID. NO:1), Gly-Pro-Gly-Gly (SEQ IDNO:2), Val-Ala-Pro-Gly (SEQ ID NO:3), Arg-Pro-Lys-Pro (SEQ ID NO:4), andArg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetOH (SEQ ID NO:5).

The invention further encompasses methods for modulating apoptosis inhuman pluripotent cell populations comprising modifying expression of anapoptotic regulating factor, and/or modulating the intracellularconcentration of endogenous lipid second messengers, such as ceramide.Preferably, the apoptotic regulating factor is PAR-4.

In another preferred embodiment of the above methods, an embryoid bodyis formed upon culturing the pluripotent human cell or cell culture withan essentially serum free medium, wherein the serum free medium isoptionally a MEDII conditioned medium, the embryoid body is dispersed toan essentially single cell suspension, and the essentially single cellsuspension is cultured with a composition comprising the amphiphiliclipid compound until the human neural cell is produced or the human cellculture enriched in neural cells is produced. In another embodiment, theembryoid body is formed upon culturing the pluripotent human cell orcell culture with a medium, the embryoid body is dispersed to anessentially single cell suspension, and the essentially single cellsuspension is cultured with a composition comprising the amphiphiliclipid compound until the human neural cell is produced or the human cellculture enriched in neural cells is produced. In a preferred embodiment,the essentially single cell suspension is cultured with the amphiphlliclipid compound in an essentially serum free medium. In a furtherembodiment, the essentially serum free medium comprises a MEDIIconditioned medium, proline, or a proline containing polypeptide. Inother preferred embodiments, the embryoid body is subsequently culturedwith one or more cell differentiation environments to produce a humanneural cell or human cell culture enriched in neural cells, wherein eachenvironment is appropriate to the cell types as they appear from thepreceding cell type. The amphiphilic lipid compound is selected from thegroup consisting of a ceramide compound, a sphingosine compound, and ahydroxyalkyl ester compound. In a preferred embodiment, the amphiphiliclipid compound is a ceramide compound of the serinol type.

As used herein, the term “cell differentiation environment” refers to acell culture condition wherein the pluripotent cells or embryoid bodiesderived therefrom are induced to differentiate into neural cells, or areinduced to become a human cell culture enriched in neural cells.Preferably the neural cell lineage induced by the growth factor will behomogeneous in nature. The term “homogeneous,” refers to a populationthat contains more than 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% of the desired neural cell lineage.

In one embodiment, the cell differentiation environment comprises anamphiphilic lipid compound. In a preferred embodiment, the amphiphiliclipid compound is a ceramide compound. In a further embodiment, the celldifferentiation environment is a suspension culture. As used herein, theterm “suspension culture” refers to a cell culture system whereby cellsare not tightly attached to a solid surface when they are cultured.Non-limiting examples of suspension cultures include agarose suspensioncultures, and hanging drop suspension cultures. In one embodiment, thecell differentiation environment comprises a suspension culture wherethe tissue culture medium is Dulbecco's Modified Eagle's Medium andHam's F12 media (DMEM/F12), and it is supplemented with a fibroblastgrowth factor (FGF) such as FGF-2. In a preferred embodiment, the celldifferentiation environment comprises an FGF. In a preferred embodiment,the cell differentiation environment comprises a suspension culturewhere the tissue culture medium is DMEM F12, FGF-2, and MEDIIconditioned medium. In a preferred embodiment, the suspension culture isan agarose suspension culture. In preferred embodiments, the celldifferentiation environment is also essentially free of human leukemiainhibitory factor (hLIF).

In other embodiments, the cell differentiation environment can alsocontain supplements such as L-Glutamine, NEAA (non-essential aminoacids), P/S (penicillin/streptomycin), N2 supplement (5 μg/ml insulin,100 μg/ml transferrin, 20 nM progesterone, 30 nM selenium, 100 μMputrescine (Bottenstein, and Sato, 1979 PNAS 76, 514-517) andβ-mercaptoethanol (β-ME). It is contemplated that additional factors maybe added to the cell differentiation environment, including, but notlimited to fibronectin, laminin, heparin, heparin sulfate, retinoicacid, members of the epidermal growth factor family (EGFs), members ofthe fibroblast growth factor family (IGFs) including FGF2 and/or FGF8,members of the platelet derived growth factor family VDGFs),transforming growth factor (TGF)/bone morphogenetic protein (BMP)/growthand differentiation factor (GDF) factor family antagonists including butnot limited to noggin, follistatin, chordin, gremlin, cerberus/DANfamily proteins, ventropin, and amnionless. TGF, BMP, and GDFantagonists could also be added in the form of TGF, BUT, and GDFreceptor-Fc chimeras. Other factors that may be added include moleculesthat can activate or inactivate signaling through Notch receptor family,including but not limited to proteins of the Delta-like and Jaggedfamilies. Other growth factors may include members of the insulin likegrowth factor family (IGF), the wingless related (WNT) factor family,and the hedgehog factor family. Additional factors may be added topromote neural stem/progenitor proliferation and survival as well asneuron survival and differentiation. These neurotrophic factors includebut are not limited to nerve growth factor (NGF), brain derivedneurotrophic factor DNF), neurotrophin-3 (NT-3), neurotrophin-4/5(NT-4/5), interleukin-6 (IL-6), ciliary neurotrophic factor (CNTF),leukemia inhibitory factor (LIF), cardiotrophin, members of thetransforming growth factor (TGF)/bone morphogenetic protein (BMP)/growthand differentiation factor (GDF) family, the glial derived neurotrophicfactor (GDNF) family including but not limited to neurturin,neublastin/artemin, and persephin and factors related to and includinghepatocyte growth factor. Neural cultures that are terminallydifferentiated to form post-mitotic neurons may also contain a mitoticinhibitor or mixture of mitotic inhibitors including but not limited to5-fluoro 2′-deoxyuridine and cytosine β-D-arabino-furanoside (Ara-C).The cell differentiation environment can further comprise conditionsthat are known to lead to an increase in endogenous ceramide levels,including but not limited to ionizing radiation, UV light radiation,application of retinoic acid, heat shock, chemotherapeutic agents suchas but not limited to daunorubicin, and oxidative stress. Endogenousceramide levels can also be elevated by incubating the cells in mediumcontaining a sphingomyelinase or a compound with similar activity, or bytreating the cells with an inhibitor of ceramidase such asN-oleoylethanolamine.

In another embodiment, the cell differentiation environment can containcompounds that enhance the activity of the amphiphilic lipid compound.In an alternative embodiment, the cell differentiation environment cancontain other inducers or enhancers of apoptosis that synergize with theactivity of the amphiphilic lipid compounds. In a further embodiment,the cell differentiation environment can comprise compounds that makethe neural cells more resistant to apoptosis. In this embodiment, theaddition of compounds that increase the resistance of neural cells toamphiphilic lipid compound enhanced apoptosis allows for the use ofhigher levels of the amphiphilic lipid compounds. As used herein, theterm “higher levels” refers to concentrations of the amphiphilic lipidcompound that would inhibit the growth or differentiation of neuralcells in the absence of the additional compound, but that do not inhibitthe growth or differentiation in the presence of the additionalcompound.

In other embodiments, the cell differentiation environment comprises anadherent culture. As used herein, the term “adherent culture” refers toa cell culture system whereby cells are cultured on a solid surface,which may in turn be coated with a substrate. The cells may or may nottightly adhere to the solid surface or to the substrate. The substratefor the adherent culture may further comprise any one or combination ofpolyornithine, laminin, poly-lysine, purified collagen, gelatin,extracellular matrix, fibronectin, tenacin, vitronectin, poly glycolyticacid (PGA), poly lactic acid (PLA), poly lactic-glycolic acid (PLGA) andfeeder cell layers such as, but not limited to, primary astrocytes,astrocyte cell lines, glial cell lines, bone marrow stromal cells,primary fibroblasts or fibroblast cells lines. In addition, primaryastrocyte/glial cells or cell lines derived from particular regions ofthe developing or adult brain or spinal cord including but not limitedto olfactory bulb, neocortex, hippocampus, basal telencephalon/striatum,midbrain/mesencephalon, substantia nigra, cerebellum or hindbrain may beused to enhance the development of specific neural cell sub-lineages andneural phenotypes.

In other embodiments of the present invention, it is not required thatan embryoid body is formed upon culturing the pluripotent human cell orcell culture. In these embodiments, a pluripotent human cell or cellculture is cultured with a medium, and as an additional step, theresultant cells are cultured with a composition comprising anamphiphilic lipid compound to produce a human neural cell or human cellculture enriched in neural cells. In some embodiments, prior toculturing the cell with the composition comprising the amphiphilic lipidcompound, the pluripotent human cell is first cultured with anessentially serum free medium. In other embodiments, the essentiallyserum free medium is a MEDII conditioned medium or the bioactivecomponent of a MEDII conditioned medium. In still other embodiments, thecells cultured with the amphiphilic lipid compound are subsequentlycultured with one or more cell differentiation environments to produce ahuman neural cell or human cell culture enriched in neural cells,wherein each medium is appropriate to the cell types as they appear fromthe preceding cell type. In a preferred embodiment, the amphiphiliclipid compound is selected from the group consisting of a ceramidecompound, a sphingosine compound, and a hydroxyalkyl ester compound. Ina preferred embodiment, the amphiphilic lipid compound is a ceramidecompound of the β-hydroxyalkylamine type.

The present invention further contemplates methods of enhancing theefficiency of the transplantation of a cultured human pluripotent cellor cell culture, comprising the steps of (a) culturing a humanpluripotent cell with a growth medium comprising a ceramide compound ofthe general formula described above, wherein R is a saturated or mono-or polyunsaturated (cis or trans) alkyl group having greater than 2carbon atoms, and R1, R2, R3, and R4 may be the same or different andare saturated or mono-or polyunsaturated hydroxylated alkyl groups, arylgroups, or hydrogen; and (b) transplanting the cultured humanpluripotent cell or cell culture into the patient. In one embodiment, R4is an alkyl chain having from 1 to 12 carbon atoms. In a preferredembodiment, R is a saturated or mono- or polyunsaturated (cis or trans)alkyl group having from 12-20 carbon atoms, the hydroxylated alkylgroups have from 1-6 carbon atoms, and R1 and R2 are hydroxylated alkylgroups. In other preferred embodiments, the ceramide compound isselected from the group comprising S16, S18 and functional homologues,isomers, and pharmaceutically acceptable salts thereof. In a preferredembodiment the ceramide compound is S18. In another preferred embodimentthe ceramide compound is S16. In a preferred embodiment of the abovemethod, the cell population comprising the cultured human pluripotentcell contains at least 80% of a neural cell.

As used herein, the term “neural cell” includes, but is not limited to,a neurectoderm cell; an EPL derived cell; a glial cell; a neural cell ofthe central nervous system such as a dopaminergic cell, a differentiatedor undifferentiated astrocyte or oligodendrocyte; a neural stem cell, aneural progenitor, a glial progenitor, an oligodendrocyte progenitor,and a neural cell of the peripheral nervous system. As used herein, theterm “neurectoderm” refers to undifferentiated neural progenitor cellssubstantially equivalent to cell populations comprising the neural plateand/or neural tube; or a partially differentiated neural progenitorcell. Neurectoderm cells are multipotential. Therefore, “neural cell” asused in the context of the present invention, is meant that the cell isat least more differentiated towards a neural cell type than thepluripotentcell from which it is derived. Also as used herein, producinga neural cell encompasses the production of a cell culture that isenriched for neural cells. In preferred embodiments, the term “enriched”refers to a cell culture that contains more than approximately 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the desired cell lineage.

The present invention further contemplates a composition for promotingmaintenance, proliferation, or differentiation of a human neural cell,the composition comprising a cell culture medium comprising MEDIIconditioned medium or the bioactive component of a MEDII conditionedmedium and an amphiphilic lipid compound of the general formulasdescribed above. Preferably the amphiphilic lipid compound is selectedfrom the group consisting of the ceramide compound, the sphingosinecompound, and the hydroxyalkyl ester compound of the formulas describedabove. In a preferred embodiment, the amphiphilic lipid compound is aceramide compound of the β-hydroxyalkylamine type, wherein R is asaturated or mono- or polyunsaturated (cis or trans) alkyl group havingfrom 12-20 carbon atoms, the hydroxylated alkyl groups have from 1-6carbon atoms, and R1 and R2 are hydroxylated alkyl groups. In oneembodiment, the ceramide compound is selected from the group consistingof N-(2-hydroxy-1-(hydroxymethyl)ethyl)-palmitoylamide (“S16”);N-(2-hydroxy-1-(hydroxymethyl)ethyl)-oleoylamide (“S18”);N,N-bis(2-hydroxyethyl)palmitoylamide (“B16”);N,N-bis(2-hydroxyethyl)oleoylamide (“B18”);N-tris(hydroxymethyl)methyl-palmitoylamide (“T16”);N-tris(hydroxymethyl)methyl-oleoylamide (“T18”); N-acetyl sphingosine(“C2”); D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol(“D-threo-PDTP”);D-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol(“D-Threo-PPMP”); D-erythro-2-tetradecanoyl-1-phenyl-1-propanol(“D-MAPP”); D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (“KAPP”);and N-hexanoylsphingosine (C6-ceramide). In more preferred embodiments,the ceramide compound is selected from the group comprising S16, S18 andfunctional homologues, isomers, and pharmaceutically acceptable saltsthereof. In a preferred embodiment the ceramide compound is S18. Inanother preferred embodiment the ceramide compound is S16. In otherembodiments, the amphiphilic lipid compound is a sphingosine compound,wherein the sphingosine compound is selected from the group consistingof D-erythro-sphingosine, L-threo-sphingonine, dimethylsphingosine, andN-oleoyl ethanolamine. In other embodiments, the amphiphilic lipidcompound is a hydroxyalkyl ester compound, wherein the hydroxyalkylester is laurylgallate. The composition comprising the amphiphilic lipidcompound may further comprise pharmaceutically acceptable carriers,excipients, additives, preservatives, and buffers. The invention alsocontemplates the neural cell or human cell culture enriched in neuralcells that is cultured in the composition.

The MEDII conditioned medium described herein can comprise one or morebioactive components selected from the group consisting of a lowmolecular weight component; a biologically active fragment of any of theaforementioned proteins or components; and an analog of any of theaforementioned proteins or components. The bioactive component is aneural inducing factor, and in a preferred embodiment, is isolated fromMEDII conditioned medium using purification techniques well known in theart. At each step of the purification procedure the samples or fractionsare applied the pluripotent cell to test for the presence of the neuralinducing factor. The bioactive component can be proline or a prolinecontaining peptide

The step of culturing the human pluripotent cells with the MEDIIconditioned medium to produce embryoid bodies (EBs) or EPL cells can beconducted in any suitable manner. For example, EPL cells may begenerated in adherent culture or as cell aggregates in suspensionculture. EBs may be generated in suspension culture using the hangingdrop technique or by culturing the cells on agarose coated plates. EBscan be generated in serum containing medium, or in essentially serumfree medium. It is also to be understood that the step of culturing theembryoid body with an essentially serum free medium and/or anessentially serum free cell differentiation environment can also beconducted in any manner known to those of skill in the art. In oneembodiment, the embryoid body is initially generated in serum containingmedium and then transferred to an essentially serum free medium forfurther neural differentiation and ceramide treatment.

As stated above, the present invention provides a method of producing aneural cell or producing a human cell culture enriched in neural cellscomprising the steps of: a) providing a pluripotent human cell; b)culturing the pluripotent human cell with an essentially serum freeMEDII conditioned medium to form an embryoid body, and c) culturingcells from the embryoid body with a composition comprising a ceramidecompound to produce the neural cell or the human cell culture enrichedin neural cells. It is to be understood that the step of culturing thepluripotent cell with the essentially serum free MEDII conditionedmedium can include the use of a “normal” or “other” essentially serumfree medium supplemented with a RMDII conditioned medium The “normal” or“other” medium, such as a normal human ES medium, can be supplementedwith an essentially serum free MEDII conditioned medium at anyconcentration, but it is preferred that the “normal” or “other” mediumcan be supplemented at between approximately 10-75%, more preferablybetween approximately 40-60% and most preferably approximately 50%essentially serum free MEDII conditioned medium. The “normal” or “other”medium that is supplemented with essentially serum free MEDIIconditioned medium is also essentially serum free, containing no oressentially no serum. In one embodiment, the pluripotent human cell iscultured with the essentially serum free cell differentiationenvironment between approximately 1-60 days, more preferably betweenapproximately 2-28 days, and most preferably 5-15 days.

The present invention encompasses the human neural cells and the humancell cultures enriched in neural cells produced by any of theabove-described methods. In preferred embodiments, the neural cell iscapable of expressing one or more of the detectable markers for tyrosinehydroxylase (T), vesicular monamine transporter (VMAT) dopaminetransporter (DAT), and aromatic amino acid decarboxylase (AADC, alsoknown as dopa decarboxylase). In preferred embodiments, the neural cellexpresses less Oct-4 protein than an embryonic stem cell or apluripotent human cell. The human neural cells or cell cultures enrichedin neural cells generated using the compositions and methods of thepresent invention can be generated in adherent culture or as cellaggregates in suspension culture. Preferably, the human neural cells orcell cultures enriched in neural cells are produced in suspensionculture.

The present invention further comprises the use of cell sortingtechniques at any one or more stage of any of the above-describedmethods. In certain embodiments, the cell sorting techniques compriselabeling the cell population and subsequent selecting cells which haveor have not been labeled. The term “label” refers to a molecule orcomposition of molecules that is detectable by optical, spectroscopic,photochemical, biochemical, immunological, chemical or magnetic means.Labels can be specifically targeted to selected cells, but need not be.Such markers or labels include, but are not limited to, colored,radioactive, fluorescent, ultraviolet, or magnetic molecules orparticles conjugated to antibodies or other molecules or particles knownto bind to cells or cellular components. Antibodies are often used aslabel components because of their ability to target specific cell types.Other reactive label components that can serve as alternatives toantibodies include, but are not limited to, genetic probes, dyes,fluorochromes, proteins, peptides, amino acids, sugars, polynucleotides,enzymes, coenzymes, cofactors, antibiotics, steroids, hormones orvitamins. The label often generates a measurable signal, which can bedetected with or without some kind of stimulatory event and can be usedto detect the presence of bound label and possibly quantitate the amountof bound label in a sample. Furthermore, the label may be a detectableintrinsic property of the cell, such as cell size or morphology, whichis detectable, for example, by measuring light scatteringcharacteristics. The label may be directly detectable or indirectlydetectable or operate in conjunction with another label. For furtherexamples of labels see those listed in Handbook of Fluorescent Probesand Research Chemicals, 9th Ed., Molecular Probes, Inc., Eugene, Oreg.In one embodiment, the protein is labeled with an antibody. In oneembodiment, the cell sorting technique comprises the use of fluorescenceactivated cell sorting, or FACS to separate the labeled cells from thenon-labeled cells. Other cell sorting techniques are well-known to thoseof ordinary skill in the art, and may be employed in the methods of thecurrent invention.

The human neural cells produced using the methods of the presentinvention have a variety of uses. In particular, the neural cells can beused as a source of nuclear material for nuclear transfer techniques,and used to produce cells, tissues or components of organs fortransplant. The invention contemplates that the neural cells of thepresent invention are used in human cell therapy or human gene therapyto treat a patient having a neural disease or disorder, including butnot limited to Parkinson's disease, Huntington's disease, lysosomalstorage diseases, multiple sclerosis, memory and behavioral disorders,Alzheimer's disease, epilepsy, seizures, macular degeneration, and otherretinopathies. The cells can also be used in treatment of nervous systeminjuries that arise from spinal cord injuries, stroke, or other neuraltrauma or can be used to treat neural disease and damage induced bysurgery, chemotherapy, drug or alcohol abuse, environmental toxins andpoisoning. The cells are also useful in treatment of peripheralneuropathy such as those neuropathies associated with injury, diabetes,autoimmune disorders or circulatory system disorders. The cells may alsobe used to treat diseases or disorders of the neuroendocrine system, andautonomic nervous system including the sympathetic and parasympatheticnervous system. In a preferred embodiment, a therapeutically effectiveamount of the neural cell or cell culture enriched in neural cells isadministered to a patient with a neural disease. As used herein, theterm “therapeutically effective amount” refers to that number of cellswhich is sufficient to at least alleviate one of the symptoms of theneural disease, disorder, nervous system injury, damage or neuropathy.In a preferred embodiment, the neural disease is Parkinson's disease.

The neural cells of the invention can also be used in testing the effectof molecules on neural differentiation or survival, in toxicity testingor in testing molecules for their effects on neural or neuronalfunctions. This could include screens to identify factors with specificproperties affecting neural or neuronal differentiation, development,survival, plasticity or function. In this application the cell culturescould have great utility in the discovery, development and testing ofnew drugs and compounds that interact with and affect the biology ofneural stem cells, neural progenitors or differentiated neural orneuronal cell types. The neural cells can also have great utility instudies designed to identify the cellular and molecular basis of neuraldevelopment and dysfunction including but not limited to axon guidance,neurodegenerative diseases, neuronal plasticity and learning and memory.Such basic neurobiology studies may identify novel molecular componentsof these processes and provide novel uses for existing drugs andcompounds, as well as identify new drug targets or drug candidates.

The neural cell or the human cell culture enriched in neural cells maydisperse and differentiate in vivo following brain implantation. Inparticular, following intraventricular implantation, the cell can becapable of dispersing widely along the ventricle walls and moving to thesub-ependymal layer. The cell can be further able to move into deeperregions of the brain, including into the untreated (e.g., by injection)side of the brain into sites that include but are not limited to thethalamus, frontal cortex, caudate putamen and colliculus. In additionthe neural cell or human cell culture enriched in neural cells can beinjected directly into neural tissue with subsequent dispersal of thecells from the site of injection. This could include any region,nucleus, plexus, ganglion or structure of the central or peripheralnervous systems. In a preferred embodiment, following brainimplantation, the neural cell or the human cell culture enriched inneural cells previously cultured with the ceramide compound induces theformation of fewer teratomas than cells or cell cultures not culturedwith the compound.

The method of enriching populations of stem or progenitor cells viaceramide induced cell death has potential applications in other areas aswell. For example, autologous transplants of hematopoietic stem orprogenitor cells may be useful in the treatment of cancers including butnot limited to cancers of the hematopoietic system such as leukemias andlymphomas as well as solid tumors. To date, this approach has hadlimited success due to the infusion of cancerous cells along with normalhematopoietic cells in the autologous graft (Rill et al., 1994 Blood,84:380-383). Efforts directed at removing-cancer cells from autologousgrafts of hematopoietic cells by cell sorting protocols have not yetbeen uniformly successful in completely removing cancerous cells fromthe autografts resulting in the potential or actual recurrence ofdisease in recipients of the autologous hematopoietic graft (Dreger etal., 2000 Experimental Hematology, 28:1187-1196; Rasmussen et al., 2002Experimental Hematology, 30:82-88). Incubation of hematopoietic cellswith ceramide analogs or the activation of ceramide signaling pathwaysin these cell populations may remove cancerous or tumor forming cellswithin these populations.

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains. The followingexamples are not intended to limit the scope of the claims to theinvention, but are rather intended to be exemplary of certainembodiments.

EXAMPLES Example 1

Production of Ceramide Analogs

Ceramide analogs were produced as described in U.S. Pat. No. 6,410,597to Bieberich, the entire contents of which are hereby incorporated byreference. Briefly, the compound S16(N-(2-hydroxy-1-(hydroxymethyl)ethyl)-paimitoylamide) was synthesizedfrom a solution of 50 mg (549 μmoles) of 2-amino-1,3-propanediol in 15ml of pyridine supplemented with 1.65 mmol (457 μl) of palmitoylchlorideat −30° C. The reaction mixture was stirred for 2 hours at roomtemperature followed by the addition of 30 ml of CH₃OH. After stirringfor another 2 hours at room temperature the reaction mixture wasconcentrated by evaporation. For selective hydrolysis of any estergroups formed during the reaction, the concentrate was treated with a 30ml solution of CH₃OH and sodium methoxide (pH 11-12) and stirred for 2hours at room temperature. The reaction mixture was neutralized withdilute HCl and then concentrated. The reaction product obtained waspurified by chromatography on a silica gel column (5 g) with CHCl₃/CH₃OH(5:1 by volume) as the eluent The yield of S16 was 135 mg (75%). Thepurity and structure were verified by nuclear magnetic resonance (NMR)and mass spectrometry.

The octanoyl-, oleoyl-, and stearoyl derivatives (S8, S18 and SS18) weresynthesized following the procedure used above for the synthesis of S16,but using octanoyl chloride, oleoyl chloride and stearoyl chloride,respectively, instead of palmitoyl chloride in the procedure.

The T16 compound was prepared by following the procedure used above forthe synthesis of S16, but using bishydroxyethyl)amine instead of2-amino-1,3-propanediol. The T18 was prepared by following the procedureused above for the synthesis of T16, but using oleoyl chloride insteadof palmitoyl chloride in the procedure.

The ceramide compounds were lyophilized and stored in the dark untiluse. The compound was dissolved in ethanol to make a stock solution, andthe stock solution was added to an appropriate pre-warmed tissue culturemediumprior to culturing the cells with the ceramide compound.

Example 2

Production of Essentially Serum Free MEDII Conditioned Medium, andIsolation of Small Molecular Weight Component of MEDII Media

Serum free MEDII (sfMEDII) was used as a source of the biologicallyactive factor in all purification protocols. An essentially serum freeMEDII conditioned medium was produced as follows. Hep G2 cells (Knowleset al., 1980 Nature, 288:615-618; ATCC HB-8065) were seeded at a densityof 5×10⁴ cells/cm² and cultured for three days in DMEM. Cells werewashed twice with 1× PBS and once with serum free medium (DMEMcontaining high glucose but without phenol red, supplemented with 1 mML-glutamine, 0.1 mM β-ME, 1× ITSS supplement (Boehringer Mannheim), 10mM HEPES, pH 7.4 and 110 mg/L sodium pyruvate) for 2 hours. Fresh serumfree medium was added at a ratio of 0.23 ml/cm² and the cells werecultured for a further 34 days. SfMEDII was collected, sterilized andstored.

Large Scale Preparation of R and E Fractions from sfMEDII

The starting material for purification and analysis of bioactive factorsfrom MEDII was derived by ultrafiltration of sfMEDII over an AmiconDiaflo YM3 membrane using a 400 ml ultrafiltration cell (Amicon) at 4°C. under nitrogen pressure. The retained fraction (R), >3×10³M_(r), wasused immediately or aliquoted and stored at −20° C. The eluted fraction(E), <3×10³M_(r), was used immediately or stored at 4° C.

Purification of the Low Molecular Weight Component of the EPLCell-Inducing Activity

220 ml of E was applied to a Sephadex G10 column (1100 ml bed volume,110×113 mm) equilibrated in water. Elution was with water at roomtemperature at a flow rate of 35 ml/minute. Fractions of 45 ml werecollected and a 1 ml aliquot of each fraction was lyophilized.Lyophilized fractions were resuspended in 100 μl of water and 25 μl wasassayed for neural and/or EPL cell-inducing activity. Activity wasdetected in fractions 6-10, 19-25.2 minutes after injection.

Fractions 7 to 9 were pooled, lyophilized and resuspended in 1 ml of30:70 methanol:acetonitrile. Samples were centrifuged at 14,000 rpm for10 minutes to remove precipitates and applied to a 10 mm Waters radialpak normal phase silica column (8 mm I.D.) attached to a Waters 510 HPLCmachine. The column was washed with 30:70 methanol: acetonitrile at aflow rate of 0.2 ml/minute for 15 minutes before the material was elutedwith a 20 minute linear gradient against water using a flow rate of 0.5ml/minute. Eluted material was detected with a Waters 490E programmablemultiwavelength detector set at 215 nm. One ml fractions were collected,lyophilized, resuspended in 50 μl DMEM and assayed for neural and/or EPLcell inducing activity which eluted from the column at 70% water/30%(30:70 methanol:acetonitrile).

The fractions of highest activity from normal phase chromatography,between 32 and 35 minutes, were lyophilized, resuspended in 50 μl waterand 10 μl was applied to a Superdex peptide gel filtration column(Pharmacia) connected to a SMART micropurification system (Pharmacia)and equilibrated in water at room temperature. The column was elutedwith water at a flow rate of 25 μl/minute and 25 μl samples werecollected. This was repeated 5 times to obtain adequate sample foranalysis. Individual samples were assayed directly for bioactivity whichwas detected in fractions eluting approximately 71.04 to 74.04 minutesafter injection in a single peak or several closely eluting peaks (i.e.fractions 8, 9 and 10). The predicted molecular weight of the activefractions was <700D according to the elution volume.

Characterization of the Purified Low Molecular Weight Component

Fraction 9 from the Superdex peptide gel filtration column waslyophilized, derivatized with FMOC and OPA and amino acid analysis wasconducted with and without hydrolysis using a Hewlett-PackardAmino-Quant II analyzer. Results were compared with a control sample ofnon-conditioned medium subjected to an identical purification. The aminoacid alanine and the amino acid proline were present in abundancecompared to the control in both hydrolyzed and unhydrolyzed samples.This indicates that these amino acids were present within the purifiedsample as free amino acids and not as peptides.

Further explanation of the small molecular weight component can be foundin International Application No. WO 99/53021, herein incorporated byreference in its entirety.

Example 3

Induction of Apoptosis by Treatment of Murine ES Cells with NovelCeramide Analogs of the β-Hydroxyalkylamine Type

Methods

In Vitro Neural Differentiation of Murine ES Cells

In vitro neural differentiation of mouse ES cells (ES-J1, ES-D3)followed a serum deprivation protocol as described previously (Hancock,et al., 2000 Biochem. Biophys. Res. Commun., 271: 418-421). Thedifferentiation stages are outlined in FIG. 2. Briefly, ES cells weregrown on gamma-irradiated feeder fibroblasts for four days in KnockoutDMEM/15% Knockout serum replacement, supplemented with ESGRO (LIF;Chemicon; Cat No. ESG1106) at a concentration of 10³ units/ml medium. EScells were then grown for another four days on gelatin-coated bacterialculture dishes without a fibroblast feeder layer, and were then grownfor three days in Knockout DMEM/15% heat-inactivated ES qualified FetalBovine Serum, supplemented with 10³ units LIF per ml of medium. Upontrypsinization, ES cells were transferred to bacterial culture disheswithout gelatin, and embryoid body (EB) formation was induced for fourdays in Knockout DMEM/10% heat-inactivated ES qualified FBS without LIF(EB4 stage). On the fifth day, floating and loosely attached EBs wererinsed off and transferred to tissue culture dishes. The EBs wereallowed to attach to the tissue culture dish surface by incubation foranother 24 hours in Knockout DMEM with 10% heat-inactivated ES qualifiedfetal bovine serum. Neural differentiation due to serum deprivation wasinduced by cultivation of the EBs for three days in DME/12 (50/50),supplemented with 1×N2 (Invitrogen/Life Technologies; Cat No. 17502,dilution of 1:100) but without serum (EB8 stage). Serum-deprived EBswere then trypsinized, plated on poly-L-ornithine/laminin-coated tissueculture dishes and grown for four days in DMEM/F12 (50/50), supplementedwith N2 and 10 ng/ml FGF-2, but without serum. This incubation period isreferred to as neuroprogenitor (NP) stage due to commitment ofneuroepithelial precursor cells to neuroprogenitor cells, and/or due tothe selective expansion of neural progenitor cells in the FGF-2containing serum-free medium. These cells have committed during the EBstages and were expanded during the NP stage. NPs grown for 48 hoursupon replating of trypsinized EBs were referred to as the NP2 stage. Onthe fifth day of NP formation, the medium was changed to Neurobasal(Invitrogen/Life Technologies; Cat No. 21103-049), with 5%heat-inactivated FBS, and the cells were incubated for another sevendays. During this time, NPs fully differentiate to glial cells andneurons. Cells cultured for 24 hours or 96 hours upon changing themedium were referred to as the D1 or D4 stage, respectively.

ES cells were cultured and differentiated to the EB4, EB8, NP2, or D4stage following the protocol as described above. The ceramide analog S18was dissolved in ethanol at a concentration of 100 mM and then added tothe cells at a final concentration of 75 μM in medium. The cells at theEB8 stage were incubated for 48 hours in the presence of the ceramideanalog and were then transplanted into mouse brains.

Ceramide Analysis

The extraction and quantitative determination of the ceramide levels byhigh performance thin layer chromatography (HPTLC) followed a standardprotocol as described previously (Bieberich et al., 2001, J. Biol.Chem., 276:44396-44404; and Bieberich et al., 1999, J. Neurochem.,72:1040-1049). Briefly, ES cells and ES-derived neural cultures werehomogenized in 500 μl of deionized water and lipids were extracted with5 ml of CHCl₃/CH₃OH (1:1 by volume). The lipid extract was adjusted tothe composition of solvent A (CHCl₃/CH₃OHMH₂O, 30:60:8 by volume) andacidic and neutral lipids were separated by chromatography on 1 ml ofDEAE-Sephadex A-25. The unbound neutral lipids were washed out with 6 mlof solvent A and were then concentrated by evaporation with a gentlestream of nitrogen. The dried residue was re-dissolved in methanol forseparation by HPTLC using the running solvent CHCl₃/HOAc(methanol:acetic acid; 9:1 by volume). Lipids were stained with 3%cupric acetate in 8% phosphoric acid for quantification by comparisonwith various amounts of standard lipids.

Imunofluorescence Microscopy and TUNEL Assay

Differentiating ES cells were grown on cover slips and fixed with 4%paraformaldehyde in phosphate-buffered saline (PBS). Fixed cells werepermeabilized with 0.5% Triton X-100 in PBS for 5 minutes at roomtemperature and unspecific binding sites were saturated by incubationwith 3% ovalbumin in PBS for 1 hour at 37° C. The cover slips were thenincubated with 5 μg/ml primary antibody (anti-ceramide clone 15B4 mouseIgM, Alexis; anti-PAR-4 rabbit IgG, Santa Cruz; anti-PCNA rabbit IgG,Santa Cruz; anti-nestin clone 401 rat IgG, BD Pharmingen) in 0.1%ovalbumin/PBS, followed by incubation with the appropriatefluorescence-labeled secondary antibody (5 μg/ml Alexa 546 conjugatedanti-mouse IgG, Molecular Probes; Alexa 488 conjugated anti-rabbit IgG,Molecular Probes, Cy3 conjugated anti-mouse IgM, Jackson) for 2 hours at37° C. The nuclei were stained by treatment with 2 μg/ml Hoechst 33258in PBS for 30 minutes at room temperature. Apoptotic nuclei were stainedusing the fluorescein FragEL TUNEL assay (Oncogene) according to themanufacturer's instructions.

Statistical Analysis

Antigen specific immunostaining was quantified by counting cells thatfluoresced at least twice as much as the background fluorescence. Cellcounts were performed in five areas of approximately 200 cells each thatwere obtained from three independent immunostaining reactions. A Chisquare test with one degree of freedom was applied for the statisticalanalysis of the distribution of two immunostained antigens. The firstnull hypothesis (HO¹) to be refuted was that the two antigens wereindependently distributed within the total cell population (mean of 200cells in five counts). The expected frequency for double-staining wasthe frequency product for immunostaining of A or B in the totalpopulation, f(A and B)=f(A)×f(B3). The second null hypothesis (HO²) tobe refuted was that the frequency of antigen B in the subpopulation Awas identical to its frequency in the total population, f(B in A)=f(B3in A+B).

Results

The concentration of endogenous ceramide in apoptotic, undifferentiatedstem cells and non-apoptotic, neural progenitor cells was determined. Incontrast to cancer cells, the undifferentiated stem cells and neuralprogenitor cells had elevated levels of endogenous ceramide prior totreatment with the ceramide compounds, indicating that ceramide analogsof the serinol type enhance or sustain apoptosis in undifferentiatedstem cells, rather than inducing or initiating apoptosis in theundifferentiated stem cells. However, neural progenitor cells, althoughthey had elevated levels of endogenous ceramide, were protected againstceramide compound-induced and/or -enhanced apoptosis.

The degree of apoptosis that occurred naturally in differentiating mouseES cells or that occurred upon incubation for 15 hours with 75 μM of thenovel ceramide analogs S16 or S18, or 35 μM N-acetyl sphingosine(C2-ceramide) was determined. FIG. 2 shows the in vitro neuraldifferentiation of mouse embryonic stem cells, indicating the variousstages of differentiation. FIGS. 3 and 4 show that cell death wasprominent at the EB8 or NP2 stages, whereas differentiated neurons didnot reveal characteristics of apoptotic cells. The degree of apoptosiswas quantified by counting TUNEL stained (apoptotic) cells. Apoptosiswas elevated at the EB8 stage, when 20±5% of cells were apoptotic, andwas most prominent at the NP2 stage when 35±5% of cells were apoptotic.Incubation with S16, S18, or C2-ceramide enhanced apoptosis, andincreased the number of TUNEL stained cells to 45±10% at the EB8 stageand 70±10% at the NP2 stage. Enhancement of apoptosis by ceramideanalogs was also observed in undifferentiated ES cells, where 40±10% ofcells were apoptotic, and at the EB4 stage, where 25±5% of cells wereapoptotic.

The sensitivity of differentiating NP cells rapidly decreased upon thepost-treatment plating of trypsinized EBs at day 8 (EB8). Sensitivity toceramide analogs was highest for NP2, while the sensitivity to theanalogs was already less than 20% at the D1 stage. TUNEL stainingrevealed that differentiated neurons at the D4 stage did not showsignificant levels of apoptosis (<10±5%) upon incubation with ceramideanalogs.

FIG. 4F shows that at the EB8 stage, a rim of cells surrounding thecentral embryoid body resisted apoptosis induced by novel ceramideanalogs. Immunostaining of EBs with an antibody against nestin, a markerprotein for neural progenitor cells, revealed that this rim ofnon-apoptotic cells strongly stains for nestin (FIG. 5B). Therefore,neural progenitor cells that express nestin were less sensitive towardceramide induced or enhanced apoptosis, whereas nestin-negative,undifferentiated cells were sensitive to ceramide-enhanceable apoptosis.Cell counts revealed that of TUNEL positive cells, 8% were nestinpositive (5/65) while 80% (108/135) of the TUNEL negative cellsexpressed nestin protein.

A quantitative determination of different marker proteins and TUNELstaining for apoptotic cells showed that predominantly nestin negative,proliferating cell nuclear antigen (PCNA) positive cells underwentapoptosis (FIG. 6). PCNA is a specific marker protein for cells thatundergo rapid cell division. PCNA positive cells are not neuralprogenitor cells, but show rapid proliferation. These highlyproliferative cells are likely to be residual pluripotent stem cellssince these cells are known to have a cell cycle with greatlyabbreviated G1 and G2 phases while differentiated cells derived frompluripotent stem cells have longer cell cycles with longer G1 and G2phases (WO 01/23531, herein incorporated by reference). The eliminationof these rapidly proliferating cells by selective apoptosis will thusreduce significantly the risk of teratoma formation aftertransplantation of pluripotent stem cell-derived cells into the hosttissue.

Example 4

Injection of Ceramide Analog Treated EB-derived Stem Cells into MouseBrains

Methods

In vitro differentiating ES cells at stage EB8 were incubated for 2448hours with 75 μM S18, or 35 μM N-acyl sphingosine or other ceramideanalogs. Protein was isolated from cells incubated with S18 for 24 hoursand from untreated cells, separated by SDS-PAGE, and the expression ofOct-4 was analyzed by immunoblotting.

Prior to injection into the mouse brain, ES cells were labeled withVybrant-DiI (rhodamine fluorescence) for permanent vital staining andwere mixed with India ink in order to track the injection channel andcell migration/tissue integration. 1×10⁴ of the untreated ES-J1 cellswere injected, while 2×10⁴ of the S18-treated cells were injected inorder to control for the percentage of cells lost to apoptosis. The EScells were injected into the right brain hemisphere (bregma—1.5 mm, 1 mmlateral of central suture, 2.0 mm deep) of 8-10 day old C57BL6 miceusing a Hamilton syringe. After 7-21 days, the mice were sacrificed, thebrain isolated and fixed with 10% PBS-buffered formalin. The brains wereVibratome sectioned at 100 μm. The distribution of the injected cellswas determined by fluorescence microscopy.

In another preparation, apoptotic cells derived from S18-incubated EBswere fluorescence labeled using Annexin V or FLICA (fluorochromeinhibitors of caspases) staining. Apoptotic (fluorescence labeled) cellswere removed by fluorescence activated cell sorting (FACS) and thenon-apoptotic cells were used for injection into mouse brain or wereused for in vitro neuronal differentiation.

Results

The protein preparation from S18 treated cells demonstrated only 25% ofthe Oct-4 immunostaining found in the untreated control cells. Thisindicated that Oct-4 protein levels were suppressed, or that Oct-4expressing cells were eliminated such that a 75% decrease in Oct-4protein levels was observed after treatment with S18.

FIG. 7A shows that ten days after injection of the cells, massiveteratoma formation was found on the right side of the brain that wasinjected with untreated, control cells. However, EB8-derived cells thatwere treated with S18 did not show teratoma formation (FIG. 7B). Inanother experiment, EB8-derived cells were stained with a fluorescentmarker dye, Vybrant diI in order to track the migration and integrationof the injected cells into the recipient's brain tissue. FIGS. 8A-D showthat untreated cells formed numerous teratomas that resulted in death ofthe recipient at 8 days post-injection. S18-treated EB8-derived cells,however, did not form teratomas, migrated to the hippocampus, andintegrated into the host's brain tissue (FIGS. 8E-H). The host injectedwith the ceramide analog treated cells was killed after 21 days in orderto analyze the brain tissue. From two separate transplantationexperiments a total of 5 animals were implanted with S18 treated cells.No teratomas were detected in the animals implanted with S18 treatedcells. A total of 4 control animals were implanted with untreated cells.One of these controls died and its brain could not be analyzed, theremaining three control animals all contained teratomas formed from theinjected untreated cells.

Cultivation of non-apoptotic cells isolated by FACS showed the typicalmorphology of neuroprogenitors. Hence, FACS sorting and removal ofapoptotic cells from the S18-treated EBs prior to injection may furtherminimize the risk of teratoma formation and enrich the portion ofneuroprogenitors.

Example 5

Induction of Apoptosis in Mouse Neuroblastoma Cells

Mouse neuroblastoma (F-11) cells were incubated for 24 hours in 0.1,0.2, 0.5, or 1.0 μM of laurylgallate (Aldrich). Apoptosis was determinedby punctate staining of condensed nuclei with Hoechst 33258 (Sigma, 2μ/ml medium for 30 minutes at room temperature).

Results

At a concentration of 0.5 μM laurylgallate, 50% of the neuroblastomacells were observed to undergo apoptosis. At 1.0 μM of laurylgallate,100% of the cells had undergone apoptosis. These results indicates thatlaurylgallate is a very potent inducer of apoptosis inducer inneuroblastoma cells, and likely will enhance apoptosis isundifferentiated ES cells as well.

Example 6

Cell Culture Conditions for Human Embryonic Stem Cells

Manual Passaging of Human ES Cells

Human embryonic stem cells (HESCs) identified as BGN01 (BresaGen, Inc.Athens, Ga.) were used in this work. The HBESCs were grown in DMEM/F12(50/50) supplemented with 15% FBS, 5% knockout serum replacer(Invitrogen), 1× non-essential amino acids (NEAA; Invitrogen),L-Glutamine (20 mM), penicillin (0.5 U/ml), streptomycin (0.5 U/ml),human LIF (10 ng/ml, Chemicon) and FGF-2 (4 ng/ml, Sigma). The human EScells were grown on feeder layers of mouse primary embryonic fibroblaststhat were mitotically inactivated by treatment with mitomycin-C. Feedercells were re-plated at 1.2×10⁶ cells per 35 mm dish. The mitoticallyinactivated fibroblasts were cultured for at least 2 days prior to theplating of HESCs. The HESCS were manually passaged onto fresh fibroblastfeeder layers every 3-4 days using a fire-pulled Pasteur pipette.Briefly, the barrel of the Pasteur pipette was melted solid and drawnout to a solid needle approximately 1 cm long and approximately 25 μm indiameter, which was sequentially pressed through HESC colonies to form auniform grid of cuts. The same needle was passed under the colonies tolift them from the feeder layer. Entire plates of HESCs were harvested,then the colonies were broken into individual pieces defined by the gridby gentle pipetting using a 5 ml serological pipette. The pieces from asingle plate were split between 2 or 3 new plates that were coated withfeeder layers of mitotically inactivated mouse primary embryonicfibroblasts.

Generation of Embryoid Bodies from Cells in the Crater of an ES Colony

The colony morphology of HESCs was observed to differ from the typicallyobserved multilayered, domed colonies when HESCs were plated onto feedercells that had been freshly plated. When HESC's were plated on feedercells that were 0-6 hours old, but not on feeders that were 2 days oldor older, typical BESC colonies formed except that in the central regionof the colony a “crater” was observed. These central or crater cellsformed a monolayer of uniform cells within a ring of multilayered HESCs.This monolayer was in direct contact with the tissue culture plastic, orthe extracellular matrix that was left behind as the HESC colony hadpushed out the underlying feeder layer. HESC colonies typically displacethe underlying feeder layer as they seed and proliferate. Cells withinthe crater expressed the pluripotent marker Oct-4, although apparentlyat a reduced level compared to the surrounding ring of HESCs, indicatingthat they are a novel, partially differentiated cell type derived fromthe HESCs. This approach allowing the controlled development of craterHESC colonies occurred within 3 to 5 days and generated a uniformmonolayer of central cells, as opposed to stochastic differentiationproceeding over several weeks and leading to a complex heterogeneousculture (Reubinoff et al., 2001 Nature Biotech, 19:1134-1140).

Formation of Essentially Serum Free Embryoid Bodies

Manually passaged HESC cultures were washed once with DMEM/F12 and oncewith DMEM/F12 supplemented with 1×N2 supplement (Invitrogen).Undifferentiated HESC colonies were harvested into uniform colony piecesof approximately 10-100 cells using the manual passaging methodsdescribed above. Pieces were transferred to 15 ml tubes and washed in 10ml DMEM/F12 plus 1×N2 supplement. The pieces were left to settle, andthe medium was aspirated. The pieces were resuspended in 2.5 ml ofmedium, and transferred to suspension dishes.

Suspension dishes were prepared by coating the surface of non-tissueculture plastic Petri dishes with a layer of agarose. The agarosecoating was generated by pouring a molten solution of 0.5% agarose inDMEM/F12 medium into the Petri plates. The agarose coating wasequilibrated in DMEM/F12 medium. Suspension cultures contained 2.5 ml ofmedium for 35 mm dishes, or 10 ml of medium for 100 mm dishes.

Essentially serum free embryoid bodies were cultured in suspension forup to four weeks, with replenishment of the medium every 3-4 days. Theessentially serum free embryoid bodies were passaged every 5-7 days bycutting them into pieces with drawn out solid glass needles. Atpassaging, the embryoid bodies contained approximately 5000-10,000 cellsand were divided into 410 pieces. Essentially serum free embryoid bodiesformed in the presence of DMEM/F12 with 1×N2 and 4 ng/ml FGF-2 weretermed sfEBs, while essentially serum free embryoid bodies formed in thepresence of DMEM/F12 with 1×N2, 4 ng/ml FGF-2 and 50% MEDII were termedsfEBMs.

Essentially serum free embryoid bodies were generated from HESC cratercells by removing the feeder layer and HESCs growing on their surface.Watchmaker's forceps were used to hold the feeder layer at the side ofthe culture dish, and lifted this layer and the attached multilayeredHESC from the dish. This manipulation peeled the feeder layer and themultilayered parts of the HESC colonies off of the dish and themonolayer crater cells were left attached to the dish. Glass needleswere used to cut the crater monolayer to 50-200 cell size pieces, andlift them from the dish. These pieces were grown in suspension culturein the same serum free conditions as above (DMEM/12, 1×N2, L-Glutamine(20 mM), penicillin (0.5 U/ml), streptomycin (0.5 U/ml), 4 ng/ml FGF-2,with or without 50% MEDII).

Essentially serum free embryoid bodies were generated from proteasepassaged monolayer HESC colonies by Collagenase treatment HESC cultureswere treated with protease, and then washed with DMEM/F12 1×N2 and 4ng/ml FGF2. The monolayer colonies remained attached to the tissueculture plastic but became less tightly associated with the feederlayer. The feeder layer was removed using watchmaker's forceps as above.The monolayer HESC colonies were scraped off the dish using a glassneedle, were transferred to a 15 ml tube and washed twice with the samemedium and centrifuged (1000 rpm, 4 minutes). The HESC colonies weretransferred to suspension dishes for development as essentially serumfree embryoid bodies grown in the conditions described above (DMEM/F12,1×N2, L-Glutamine (20 mM), penicillin (0.5 U/ml), streptomycin (0.5U/ml), 4 ng/ml FGF-2, with or without 50% MEDII).

Immunostaining

For immunostaining, seeded embryoid bodies were rinsed with 1× PBS andfixed in 4% paraformaldehyde, 4% sucrose in 1× PBS for 30 minutes at 4°C. The cells were then washed in 1× PBS and stored at 4° C. Essentiallyserum free embryoid bodies in suspension were disaggregated and attachedto a glass slide using a standard cytospin approach for immunostaining(Watson, 1966 J. Lab. Clin. Med., 68:494-501). sfEBMs were washed with1× PBS and disaggregated with 0.05% typsin and gentle trituration. Thecell suspension was washed with culture medium, pelleted and resuspendedin HESC medium and 1×10⁴ cells were attached to a glass microscope slideby centrifugation at 300 g for 4 minutes using a cytospin apparatus(Heraeus Instruments GmbH). The attached cells were fixed immediatelywith 4% paraformaldehyde, and 4% sucrose in 1× PBS for 15 minutes,followed by three separate 5-minute washes in 1× PBS.

To perform immunostaining on fixed cells or cytospins, the samples werewashed in block buffer (3% goat serum, 1% polyvinyl Pyrolidone, 0.3%Triton X-100 in wash buffer) for 30 minutes, and then incubated with theappropriate dilution of the primary antibody, or combination ofantibodies for 4-6 hours at room temperature. The primary antibodieswere anti-Map2, a mouse monoclonal antibody recognizing the Map-2 a, band c isoforms (Sigma, Catalog # M4403) at a 1/500 dilution;anti-Nestin, a rabbit polyclonal antibody (Chemicon, Catalog # AB5922)at a 1/200 dilution; anti-Oct-4, a rabbit polyclonal antibody (SantaCruz, Catalog # sc-9081) at a 1/200 dilution; sheep anti-TyrosineHydroxlyase (TH) antibody (Pel-Freez, Catalog # P60101-0) at a 1/500dilution; anti-phosphoHistoneH3, a rabbit polyclonal antibody (Upstate,Catalog, # 06-570) at a 1/400 dilution; anti-SSEA4, a mouse monoclonalantibody (Developmental Studies Hybridoma Bank, Catalog # MC-813-70) ata ⅕ dilution. The cells were then washed in wash buffer (50 mM Tris-HCLpH 7.5, and 2.5 mM NaCl) 3 times for 5 minutes each wash. The cells werethen incubated for a minimum of 2 hours in secondary antibodies diluted1:1000, followed by washing in wash buffer. The secondary antibodieswere appropriate combinations of Alexa-350 (blue), -488 (green) or -568(red) conjugated goat anti-chicken, anti-rabbit, anti-sheep oranti-mouse antibodies, all available from Molecular Probes. Some sampleswere stained with 5 ng/ml DAPI to detect cell nuclei, and were thenwashed from overnight to 2 days in a large volume of wash buffer. Theslides were mounted with mounting medium and a cover slip. Slides werevisualized using either a NIKON TS100 inverted microscope or a NIKON TE2000-S inverted microscope with a Q Imaging digital camera

Example 7

Neural Differentiation of Essentially Serum Free Embryoid Bodies

HESCs were grown in suspension as embryoid bodies in essentially serumfree conditions in the presence of 50% conditioned medium from the HepG2hepatocarcinoma cell line (MEDII conditioned medium). The sfEBMs werecultured in suspension for up to 6 weeks, with passaging every 10 to 15days. Passaging was performed by using glass needles to dissect the EBsinto pieces, paying particular attention to the isolation of structuredrosette regions. Non-rosette regions were generally removed from theculture during the passaging process, although the solid material couldregenerate prior to the next passage.

Structured regions from essentially serum free embryoid bodies wereseeded onto polyornithine and laminin coated permanox slides foradherent culture and further analysis. Essentially serum free embryoidbodies (sfEBs and sfEBMs) were cut into pieces using glass needles and1-15 pieces were plated onto polyornithine/laminin coated permanoxchamber slides in the same medium used for suspension culture.Polyornithine/laminin coated slides were prepared by dilutingpolyornithine to 20 μg/ml in tissue culture grade water, coating chamberwells at 37° C. overnight, washing the wells twice with water andcoating the chamber wells with 1 μg/ml laminin at 37° C. for 2 hours toovernight. The slides were washed with water and 1× PBS prior to platingthe cells. The embryoid bodies were cultured on these slides for 2-7days.

Results

The structured rosette regions that were first observed morphologicallybetween 7-10 days after derivation are neurectoderm/neuralprecursor/neural tube cell types. The rosette regions could comprisemore than 50% of the mass of an essentially sfEBM. These structures takethe form of spherical rosettes with a distinct radial appearance andcentral cavity surrounded by a ring of cells that is 4-8 cells in width.Other morphologically distinct regions that were observed in essentiallyserum free embryoid bodies included fluid filled cysts and homogeneoussolid regions. Immunostaining of sections and cytospins demonstrated thepresence of neurons (Map2+ cells) in sfEBMs in suspension. The neuronalnetworks were intermingled with, and surrounded the rosette structures.When seeded in adherent culture, rosettes grew as circular or ovoidradial structures and were surrounded by large interconnected mats ofneurons that included many presumptive dopaminergic neurons that wereTH+.

Example 8

Reduction in the Level of Oct-4 Protein in Differentiated HESCs

The Oct-4 transcription factor is a tightly regulated marker ofpluripotency in the mouse, and expression of Oct-4 mRNA in human innercell mass and ES cultures has been confirmed (Hansis et al., 2000 Mol.Hum. Reprod., 6(11):999-1004, and Reubinoff et al., 2000 Nature Biotech,18:399-404). However, the restriction of Oct-4 protein to pluripotentcells in humans has not been examined thoroughly. Manually passaged HESCcultures containing domed or cratered colonies were stained with antiOct-4 antibodies.

It was observed that the Oct-4 protein is expressed at high levels inundifferentiated HBESCs (FIG. 9A) and that levels of the Oct-4 proteinare down-regulated following differentiation (FIG. 9B). An unexpectedcharacteristic of immunostaining in the culture systems analyzed wasthat differentiated human cells retained a reduced but detectable levelof Oct-4. However, when seeded sfEBM cultures were fixed andimmunostained, a process that maintains the morphology of a culture, thedifference between the two types of Octal expression was clearlydistinguishable. High level Oct-4 expression was only observed as brightnuclear staining in tightly packed but evenly spaced cells. Thereforeimmunostaining for Oct-4 expression during neural differentiation inembryoid bodies was a suitable assay for the presence of residualcompartments of pluripotent cells.

To monitor the persistence of pluripotent cells during sfEBMdifferentiation, essentially serum free embryoid bodies were generatedfrom domed HESC colonies or monolayer crater ES cells. The sfEBMs weregrown in suspension for 3-7 days, seeded onto polyornithine/laminincoated chamber slides, cultured for 3-5 days in the same medium andfixed for immunostaining. The presence of residual nests of pluripotentcells was demonstrated by clusters of high level Oct-4 immunostainingamongst the generalized low level of Oct-4 staining seen in theneuralized culture (FIG. 9C). The Oct-4 immunoreactivity wasnuclear-specific. High level Oct-4 expression was not associated withthe neural rosettes, which were visualized by the characteristic radialpattern of nuclei stained with DAPI (FIG. 9D). The presence of nests ofresidual pluripotent cells was still observed in sfEBMs that werecultured for over one month, with several passages specificallyattempting to purify the neural rosette material, highlighting thepersistent nature of these pluripotent cells and their implied teratomaforming potential when transplanted.

Example 9

Induction of Apoptosis by S18 Treatment of Seeded Embryoid Bodies

Treatment of EBs with S18

sfEBMs were derived from domed HESC colonies, grown in suspension for 24days with one passage, and seeded to polyornithine/laminin coatedchamber slides in DMEM/F12, supplemented with 1×N2 (Gibco), and 1% FCS.The seeded sfEBMs were treated with 6, 8 or 10 μM S18 dissolved in themedia for 36 hours. The cultures were then washed with DMEM/F12,supplemented with 1×N2, and 4 ng/ml FGF-2 and incubated for 24 hours in50% DMEM/F12, 50% MEDII, supplemented with 1×N2, and 4 ng/ml FGF-2before fixing and staining with DAPI.

Apoptosis in seeded serum free embryoid bodies was monitored bymorphological observation of cell death and DAPI staining to revealapoptotic nuclei. Apoptotic nuclei were observed as obviously fragmentedand degenerating nuclei, with small punctuate patterns of DAPI staining.Rosette regions from essentially serum free embryoid bodies insuspension were passaged further in the same medium, either withdrawingS18 or culturing the embryoid bodies for an additional 4 to 8 days inthe presence of S18. Rosette regions were then seeded ontopolyornithine/laminin coated slides for analysis of proliferation anddifferentiation to neural lineages.

Results

Prior to S18 treatment, the seeded cultures were heterogeneous andcontained extensive neural rosette structures (FIG. 10A) as well asother cell types, such as presumptive glial cells, or other unidentifiedcell types. S18 treatment induced apoptosis of a large proportion of theculture at each dosage, and this effect was observed within 24 hours oftreatment (FIGS. 10B, and 10C). No differences were observed between thedifferent doses of S18. Overall, the general morphology of the culturewas significantly affected, with a high level of cell death. The levelof cell death is dependent upon the proportion of cell rosettes at thetime of treatment. This proportion will vary, as will the level of celldeath. Cellular debris was observed surrounding the seeded sfEBM,indicating that the cell types that had proliferated away from the sfEBMwere killed. Neural rosette structures did not appear to be adverselyaffected by the S18 treatment, indicating that they were resistant tothe induction of apoptosis mediated by this ceramide analog.Morphologically normal rosettes could be observed within an otherwisegenerally apoptotic culture (FIGS. 10C, and 10D). DAPI staining ofcultures 24 hours after S18 withdrawal demonstrated that rosette cellshad maintained morphologically normal nuclei, whereas cells on theperiphery of the culture exhibited condensed nuclei, a characteristic ofapoptotic cells (Kerr et al., 1972 Cancer, 26:239-257; FIG. 10E). Thepossibility that non-rosette cells in the multilayered region of theseeded sfEBM survived S18 treatment could not be addressed by thisanalysis. The observation that morphologically normal nuclei were anindicator of viable cells was strengthened by the observation of mitoticfigures with DAPI staining, 24 hours after S18 withdrawal. This resultindicated that the cells that survived treatment with S18 were capableof proliferation.

It was observed that non-rosette cells in an embryoid body would notsurvive when they were located more than around 5 cell distances fromthe edge of an essentially serum free embryoid body. It is hypothesizedthat this is presumably due to problems with poor exchange of oxygen ortoxic waste metabolites. This degeneration of non-rosette cells appearedas regions containing degenerate nuclei as shown by DAPI staining.Rosette cells were not affected by distance from the edge of anessentially serum free embryoid body in the same manner, and healthynuclei could be observed more than 20 cell diameters from the edge.Degenerate regions were not observed in sfEBMs derived from proteasepassaged cells, or after exposure to S18, further indicating the highpurity of these neural rosette cell populations.

In summary, the S18 ceramide analog appeared to induce apoptosisefficiently in a range of different cell types in seeded serum beeembryoid bodies, and this induction appeared to be selective, withneural rosette cells appearing not to be affected. The application ofS18 to embryoid bodies thus provided a population of neural rosettecells with high purity.

Example 10

Ceramide Analog S18 Treatment of Essentially Serum Free Embryoid Bodiesin Suspension

Essentially serum free embryoid bodies (sfEBMs) were generated asdescribed in Example 6, and were exposed to S18 at different stages oftheir development in order to assess the timing of depletion of highOct-4 expressing cells, and in order to determine when neural rosettescould be selected. The sfEBMs in suspension were treated with 10 μM S18in 50% DMEM/F12, 50% MEDII, supplemented with 1×N2, Glutamine (20 mM),penicillin (0.5 U/ml), and streptomycin (0.5 U/ml) for varying amountsof time, and the sfEBMs were then evaluated histologically and byimmunocytochemistry.

Essentially serum free embryoid bodies were derived from proteasepassaged cells and grown in the presence of 50% MEDII conditionedmedium. The embryoid bodies were exposed to 10 μM S18 in the same mediumfrom day 6 to 9 after derivation. At day 9 the S18 treated sfFBMs andmatched control sfEBMs not exposed to S18 were fixed, embedded inplastic, cut to 3 micron sections and stained with DAPI to enable theprecise determination of the proportion of the total healthy nuclei ofan sfEBM that were rosette cell nuclei.

Results

It was not possible to derive sfEBMs from monolayer crater cells in thepresence of 10 μM S18. No viable embryoid bodies were observed in thesuspension culture after four days of S18 treatment, indicating thatcells resistant to the induction of apoptosis were not present at thisstage of the culture.

Conversely, sfEBMs at day 14 exhibited extensive neural rosettestructures. This material was exposed to 10 μM S18 in 50% MEDII mediumfor 2 days, followed by manual passaging, and an additional 4 days in 10μM S18 in the same medium. While 48 hours exposure to S18 did not haveovert morphological effects on the sfEBM, when the embryoid bodies weremanually passaged it was apparent that there was extensive apoptosis inthe bodies. The non-rosette regions of the sfEBM fragmented whenmanipulated and released extensive stringy material that was indicativeof genomic DNA from lysed cells. However, the rosette regions weremorphologically normal and could be separated from all other degenerateregions of the sfEBM. The rosette pieces were incubated in 10 μM S18 fora further 4 days, and the medium was then switched to 50% DMEM/F12, 50%MEDII, supplemented with 1×N2, and 4 ng/ml FGF-2 at day 20 after theinitial derivation of the embryoid bodies.

At day 21 some ceramide selected sfEBMs were seeded ontopolyornithine/laminin coated slides, cultured in the same medium for anadditional 8 days, and fixed for immunostaining. These seeded piecesdeveloped as rosette cultures and mats of neurons were observeddifferentiating from these precursors.

Other ceramide selected sfEBMs were maintained in suspension, and werecultured for an additional 25 days, until 45 days after their initialderivation. These suspension cultures were passaged once during thistime and initially proliferated at a rate similar to seeded neuralrosettes, although their growth rate slowed after around day 40 afterinitial derivation. At day 35, the selected sfEBMs in suspensionconsisted of what appeared to be essentially pure neural rosettematerial, without any obvious regions comprised of different cell types(FIGS. 11A and 11B).

The S18 selected sfEBM that were seeded at day 21 were analyzed byimmunocytochemistry with antibodies directed against Oct-4, Map2, TH andphospho-Histone H3. Staining with anti-Oct-4 indicated that no regionsof high Oct-4 expression could be detected in any of the S18 treatedsamples (FIGS. 12A and 12B), indicating that no residual nests ofpluripotent cells survived exposure to S18. The same result was seen inadditional experiments when sfEBMs were generated and treated with 10 μMS18 in suspension prior to plating. Low level Oct-4 expression wasdetected in rosettes (FIGS. 12A, 12B; FIG. 13A) and other cell typesthat were present in the cultures. While these cultures had a highproportion of rosette cells, it was clear that other cell types werepresent, such as neurons, as well as other presumed neuralized celltypes derived from the rosette precursor cells. Immunostaining withanti-Map2 (FIGS. 13B, and 13D), which recognizes a microtubuleassociated protein in the dendrites of mature neurons, demonstrated thepresence of networks of differentiated neurons associated with neuralrosettes. Staining with anti-TH, which recognizes tyrosine hydroxylase,the rate limiting enzyme in dopamine biosynthesis, demonstrated thatpresumptive dopaminergic neurons or their precursors were not ablated byexposure to 10 μM S18 (FIGS. 13C, and 13E). The histone H3 protein isphosphorylated during mitosis and is an effective marker of mitoticcells.

Seeded S18 selected sfEBMs were stained with anti-phosphoHistone H3 andDAPI (FIG. 13F). The presence of neural rosettes was indicated by theircharacteristic radial pattern. PhosphoHistone H3 expression demonstratedthat these cultures were actively proliferating at the time they werefixed (day 28 after derivation, 8 days after withdrawal of S18).PhosphoHistone H3 staining within the neural rosettes indicated thatthese precursor cells were still mitotically active after exposure toS18 and could therefore be expanded further.

sfEBMs derived from protease passaged cells exposed to 10 μM S18 fromday 6 to 9 after derivation were analyzed. In sections of control(untreated) sfEBMs, greater than 80% of the nuclei in the embryoidbodies were associated with rosettes (FIG. 14A). The rosette nuclei weregenerally elongated, in contrast to regions of smaller round nuclei thatwere not organized into rosettes. DAPI stained sections of S18 treatedsfEBMs showed marked differences from the control sections (FIGS.14B-D). The overall proportion of nuclei per measured area of sfEBM mayhave been reduced, but was generally still high However, nearly allnuclei in the treated sfEBM were elongated in appearance, and rosettestructures were still clearly present. The small round nuclei of thepresumptively non-rosette cells were very rarely noted. This indicatedthat a very pure population of neural precursor rosette cells hadsurvived the incubation with S18.

It was observed that non-rosette cells located more than around 5 celldistances from the edge of an essentially serum free embryoid body wouldnot survive. This effect was presumably due to problems with poorexchange of oxygen or toxic waste metabolites. The effect wascharacterized by regions containing degenerate nuclei as shown by DAPIstaining. However, location did not affect rosette cells in the samemanner, and healthy rosette nuclei could be observed more than 20 celldiameters from the edge of a sfEBM. Degenerate regions were not observedin sfEBMs derived from protease passaged cells, or after exposure toS18, further indicating the high purity of these neural rosette cellpopulations.

Example 11

Up-Regulation of Ceramide Expression During Neural Differentiation ofEmbryonic Stem Cells

Methods

Ceramide Analysis and Preparation of Ceramide-Containing Medium

The extraction and quantitative determination of ceramide by highperformance thin layer chromatography (HPTLC) was performed as describedin Example 3. Quantitative determination of ceramide using thediacylglycerol (DAG) kinase assay was performed according to Signorelliand Hannun (2002 Methods Enzymol., 345:275-294).

Fumonisin B1 (FB1) or myriocin-treated (ceramide-depleted)differentiating ES cells were incubated with natural ceramide. Toprepare the natural ceramide, 50 mg of EBs or NPs were resuspended in500 μl of water, and after phase separation with 500 μl of CHCl₃/CH₃OH(1:1 by vol.) the neutral lipids were recovered from the lower phase.The neutral lipids were evaporated to dryness with a gentle stream ofnitrogen and redissolved in 1 ml of CHCl₃. The solution was applied to asilicic acid gel column (0.5 g) and fatty acids and cholesterol washedout with another 15 ml of CHCl₃ (Dasgupta and Hogan, 2001 J. Lipid Res.,42:301-308). The ceramide fraction was then eluted with 20 ml ofCHCl₃/acetone (9:1, by vol.), evaporated to dryness, and the residue(approximately 6 nmoles of ceramide) dissolved in 20 μl of ethanolcontaining 2% dodecane (vol./vol.). Aliquots of 5 μl were mixed with 1ml of medium, yielding a final concentration of 1.5 μM natural ceramidefor induction of apoptosis (Ji et al., 1995 FEBS Lett., 358:211-214).

BrdU Labeling, Immunofluorescence Microscopy and TUNEL Assay

Differentiating ES cells at the EB8 cells were dissociated and grown for24 hours on laminin/ornithin-coated cover slips (NP1 stage) in DMEM/F12(50/50), supplemented with 1×N2 and 20 ng/ml FGF-2. Cells were incubatedfor 3 hours with 10 μM bromodeoxyuridine (BrdU) and the TUNEL assay orimmunostainings performed after 5 hours of incubation. Forimmunostaining, cells were fixed with 4% paraformaldehyde inphosphate-buffered saline (PBS). Fixed cells were permeabilized with0.2% Triton X-100 in PBS for 5 minutes at room temperature andimmunostaining performed as described previously (Bieberich et al., 2001J. Biol. Chem., 276:44396-44404). The nuclei were stained by treatmentwith 2 μg/ml Hoechst 33258 in PBS for 30 minutes at room temperature.Apoptotic nuclei were stained using the fluorescein FragEL TUNEL assayaccording to instructions of the manufacturer (Oncogene).

Statistical Analysis

Statistical analysis was performed as described in Example 3.

Results

Mouse ES cells were differentiated following a serum deprivationprotocol as described above. This method yielded ES-derived cellcultures highly enriched in neural cells after 25 days in culture (Okabeet al., 1996 Mech. Dev., 59:89-102, Hancock et al., 2000 Biochem.Biophys. Res. Commun,. 271:18421). Neuronal differentiation was verifiedby staining of marker proteins using immunoblotting (FIG. 15) andimmunofluorescence microscopy. ES cell differentiation was initiated byaggregating the ES cells to form embryoid bodies (EBs). The EBs wereincubated in suspension culture in serum-containing medium for four days(stages EB1-EB4, FIG. 2). The differentiating EBs were then plated ontissue culture plates and allowed to attach in serum-containing mediumfor one day and then shifted to serum free medium for three additionaldays of culture (EB5-EB8, FIG. 2). The Oct-4 protein, a marker ofpluripotent stem cells Resce and Scholer, 2001 Stem Cells, 19:271-278),was detected in the EB8 stage reflecting the presence of residualundifferentiated pluripotent stem cells within the EBs (FIG. 15, lane1). No Oct-4 expression was detectable after the dissociation andreplating of the EBs in serum-free, FGF-2-containing medium (FIG. 15,lanes 24). The serum-free conditions-did not support the proliferationof non-neural cell types, while the FGF-2 supported the robustproliferation of neural progenitor cells at stages NP1-NP4. The earlyneural precursor cell marker vimentin was only detected in EB8 and NP2(FIG. 15, lanes 1 and 2) while the NP marker nestin was detected at lowlevels at EB8 and at high levels at NP2 and D1 (FIG. 15, lanes 1-3). Theexpansion of EB8 was followed by a large increase of the number ofnestin-positive progenitor cells from about 60% at the NP2 stage, tomore than 80% at NP4. Differentiation of NPs to glial cells and neuronswas initiated by withdrawal of FGF-2 from the culture medium (stagesD1-D4 in FIG. 2) and verified by staining for the glial marker proteinGFAP and the neuronal marker proteins MAP-2 and synaptophysin (FIG. 15,lanes 3 and 4). No expression of NP markers was detected after D1 (FIG.15, lane 4). Expression of GFAP was detected at D1 and D4 while MAP-2and synaptophysin were only detected at D4, the most maturedifferentiation stage tested. It should be noted that less than 20% ofHoechst-stained cells showed neither GFAP nor MAP-2 staining, whichverifies that the portion of non-neuronal cells within the fullydifferentiated culture was negligible.

To measure ceramide levels during ES cell differentiation, 50-100 mg ofcells were harvested at different time points during differentiation.Sphingolipids were isolated using organic solvent extraction. As shownin FIGS. 16A and 16B, quantitative HPTLC and DAG kinase assay revealedthat fibroblast-free, undifferentiated ES-cells (FIG. 16A, lane 2)contained less than 0.2+/−0.1 μg ceramide/mg cell protein. After fourdays of EB formation (EB4 stage) ceramide had increased to 0.4+/−0.1 μgceramide/mg cell protein (FIG. 16A, lane 3). Endogenous ceramide wasfurther elevated to 1.0+/−0.2 μg/mg cell protein by the EB8 stage ofdifferentiation (FIG. 16A, lane 4). The increased ceramide concentrationwas maintained through the NP2 stage of differentiation (FIG. 16A, lane5). In the D1 stage of differentiation, the ceramide concentration wasfound to be reduced by 70% (0.3+/−0.1 μg ceramide/mg cell protein) anddid not change significantly after four days of differentiation (FIG.16A, D1, D2, D4, lanes 6, 7 and 8). Taken together, these resultsindicate that the peak elevation of ceramide occurs during the initialformation of NPs upon serum deprivation of EBs and during NP expansionin the presence of FGF-2.

Apoptosis During Neuroprogenitor Expansion is Dependent on CeramideElevation

The degree of apoptosis in differentiating ES cultures was determinedsince it was previously proposed that ceramide elevation inducesapoptosis in NPs (Bieberch et al., 2001 J. Biol. Chem.,276:44396-44404). As shown in FIG. 16B, the degree of apoptosis wasquantified by counting TUNEL-stained cells. Apoptosis was elevated atthe EB8 stage (−20+/−5%), and was highest at the NP2 stage (35+/−5%).The fraction of apoptotic cells rapidly decreased upon induction ofneural differentiation and was already less than 20% at D1.Differentiated neurons at D4 did not show a significant degree ofapoptosis (<10+/−5%). These results indicate that the peak time ofapoptosis coincided with the peak elevation of endogenous ceramide.Consistent with this, inhibition of ceramide biosynthesis by incubationwith 25 μM of the ceramide synthase inhibitor FB1 or 50 nM of the serinepalmitoyltransferase inhibitor myriocin for 48 hours reduced the degreeof apoptosis at the EB8 and NP2 stage by about 50%. At the NP2 stage,apoptosis proceeded in differentiating ES-cells that did not stain fornestin. In these cells, FB1 significantly reduced the degree ofapoptosis. Apoptosis was restored by the addition of 30 μM N-acetylsphingosine (C2-ceramide), 80 μM of the novel ceramide analog N-oleoylserinol (S18), or 1 μM of natural ceramide that was extracted fromdifferentiating ES cells. Taken together, these results showed thatelevation of endogenous ceramide was a prerequisite for the induction ofapoptosis in differentiating ES-cells at the NP2 stage.

Example 12

Ceramide-Induced Apoptosis is Dependent on PAR-4 Expression inNestin-Negative Cells

Methods

RNA Preparation and RT-PCR

Total RNA was prepared from differentiating stem cells using the Trizolmethod according to the manufacturer's (Life Systems) protocol. Analiquot (0.6-1.0 μg of RNA) was used for RT-PCR with the ThermoScript™RT-PCR system following the supplier's (Invitrogen) instructions. PCRwas carried out by applying 35 cycles with various amounts of firststrand cDNA template (equivalent to 0.05-0.2 μg of RNA) and 20 pmoles ofsense and antisense oligonucleotide primer. The followingoligonucleotide primer sequences and annealing temperatures were used:PAR-4 (sense, 5′ccagcgccaggaaaggcaaag3′ (SEQ ID NO:6); antisense,5′ctaccttgtcagctgcccaacaac3′ (SEQ ID NO:7); 61° C.), PKCζ (sense,5′agecacgccgtttggaaagg3′ (SEQ ID NO:8); antisense,5′acactttattcctcagggcattacacg3′ (SEQ ID NO:9); 58° C.), SPT1 (sense,5′gctaacatggagaatgcactc3′ (SEQ ID NO:10); antisense,5′cttcctccgtctgctccac3′ (SEQ ID NO:11); 53° C.), GAPDH (sense,5′gaaggtgaaggtcggagtcaacg3′ (SEQ ID NO:12); antisense,5′ggtgatgggatttccattgatgacaagc3′ (SEQ ID NO:13); 58° C.). The amount oftemplate from each sample was adjusted until PCR yielded equalintensities of amplification.

Construction of PAR-4-RFP cDNA and Transfection of EB-Derived Cells

For construction of PAR-4-REP cDNA, RT-PCR was performed with theoligonucleotide primer pair sense 5′atggcgaccggcggctatcg3′ (SEQ IDNO:14) and antisense 5′ctaccttgtcagctgcccaacaac3′(SEQ ID NO:15), usingthe first strand cDNA generated from embryoid bodies (EB8 stage) astemplate for the amplification reaction. The primers were endowed withthe restriction enzyme cleaving sites Eco47III (sense) and SaII(antisense) for ligation of the PAR-4 specific amplification productinto the multiple cloning site of HcRFP, a vector that encodes a far-redshifted variant of red fluorescent protein (Clontech). DifferentiatingES cells at the NP1 stage were transfected with the PAR-4-RFP constructusing the lipofectamine 2000 procedure according to the manufacturer'sinstructions (Invitrogen). The transfected cells were incubated in DMEMF12 (50/50), supplemented with 1×N2 and 20 ng/ml FGF-2, and the TUNELassay or immunostainings were performed after 48 hours of incubation andNP formation. For depletion of ceramide, differentiating ES cell wereincubated with 25 μM fumonisin B1 or 50 nM myriocin 48 hours prior totransfection with the PAR-4-RFP vector, and the inhibitor maintained inthe medium throughout the post-transfection period. For induction ofapoptosis, the novel ceramide analog S18 (40-100 μM), N-acetylsphingosine (C2-ceramide, 10-30 μM), or natural ceramide isolated fromEBs (0.5-1.5 μM) was added 48 hours after transfection with thePAR-4-RFP cDNA, and the degree of apoptosis quantified after 15-20 hoursusing TUNEL assays with paraformaldehyde-fixed cells as described above.

Morpholino Antisense Knockdown of Endogenous PAR-4

Endogenous expression of PAR-4 was suppressed by transfection ofdifferentiating ES cells at the NP1 stage with 2 nmoles (per well in 24wells plate) of the morpholino-based antisense oligonucleotide5′cgatagccgccggtcgccatgttcc3′(SEQ ID NO:16) (see Guo et al., 1998,Nature Med., 4:957-962; Guo et al., 2001 Brain Res, 903:13-25) followingthe instructions of the manufacturer (GeneTools) in 0.5 ml of serum-freeDMEM/F-12, 1XN2 supplemented with 20 ng/ml FGF-2. For induction ofapoptosis, the novel ceramide analog S18 (40-100), N-acetyl sphingosine(C2 ceramide, 130′ K, or natural ceramide isolated from EBs (0.5-1.5 μM)was added 48 hours after transfection with the anti-PAR-4 morpholino,and the degree of apoptosis quantified after 15-20 hours using TUNELassays with paraformaldehyde-fixed cells as described above.

Protein Isolation and Immunoblotting

The amount of protein was determined following a modified Folin phenolreagent (Lowry) assay essentially as described in Wang and Smith (1975Anal. Biochem., 63:414-417). Protein extracted with detergent wasprecipitated according to the Wessel and Fluegge method (Wessel andFlugge, 1984 Anal. Biochem, 138:141-143). SDS-PAGE was performed usingthe Laemmli method followed by immunoblotting (Laemmri, 1970 Nature,227:680-685).

Results

Expression of PAR-4, an inhibitor protein of PKCζ, has been reported tobe a prerequisite for induction of apoptosis by incubation ofdifferentiating ES cells with S18 or C2-ceramide (Bieberich et al., 2001J. Biol. Chem., 276:44396-44404). These results were confirmed byincubation of FB1-treated cells at the NP2 stage with S18, C2-ceramide,or natural ceramide that was extracted from differentiating ES cells.Apoptosis was only seen in cells that expressed PAR-4. S18 or ceramideincubation did not alter the number of PAR-4 positive cells but itrestored the degree of apoptosis to that found with cells that were notpre-treated with FB1. S18 incubation did not alter the concentration ofendogenous ceramide, indicating that ceramide elevation was not abyproduct of apoptotic cells but the cause of apoptosis. Notably, themajority of apoptotic cells (>90%) that stained for PAR-4 werenestin-negative.

To suppress PAR-4 expression, ES cells at the NP1 stage were transfectedwith a PAR-4 specific morpholino phophorodiamidate antisenseoligonucleotide (morpholino) that was designed on the basis of apreviously published antisense oligonucleotide sequence (Guo et al.,1998, Nature Med., 4:957-962; Guo et al., 2001 Brain Res., 903:13-25).Morpholinos have been shown to avoid many of the pitfalls associatedwith conventional antisense oligonucleotides and have been successfullyused for transfection of embryos and cultured cells (Morcos, 2001Genesis, 30:94-102). The number of TUNEL-stained cells in the negativecontrol (NPs 48 hours after transfection with a standard morpholino) wasequivalent to that found with untransfected cells (35%, see FIG. 16B),indicating that the degree of apoptosis was not affected due to anyunspecific effect of the morpholino. This result was consistent with theobservation that the expression level of PAR-4 in untransfected andcontrol morpholino-transfected NPs was the same (FIG. 17, lanes 1 and2). Incubation of control morpholino-transfected NPs with 80 (LM 818increased the number of apoptotic cells to that found with S18-treated,untransfected cells (more than 80%). However, transfection of NPs with aPAR-4 specific antisense morpholino reduced S18-induced apoptosis to alevel less than 30%. Consistently, transfection with the PAR-4 antisensemorpholino suppressed the expression level of PAR-4 to about 25% of thatdetected in untransfected cells (FIG. 17, lane 3).

To determine the effect of elevated PAR-4 expression, myriocin-treatedES cells were transfected at the NP1 stage with PAR-4 linked tofar-red-shifted red fluorescent protein (PAR-4-RFP; FIG. 17, lane 4).PAR-4-RFP expression by itself did not induce apoptosis in thetransfected cells. However, apoptosis was observed when transfectedcells were incubated with 40 μM S18, a concentration at which no or onlya low degree of ceramide analog-induced apoptosis occurred inuntransfected or mock (RFP)-transfected cells. Apoptosis was inducedalmost exclusively in PAR-4-RFP expressing cells, whereas the populationof cells without PAR-4-RFP remained unaffected. In summary, theseresults show that the expression and/or elevation of PAR-4 were aprerequisite for ceramide-induced apoptosis in differentiating ES cells.

Apoptosis is Induced by Simultaneous Up-Regulation of PAR-4 and CeramideBiosynthesis

To identify the genes involved in the regulation of apoptosis indifferentiating embryonic stem cells, the temporal expression pattern ofpro- or anti-apoptotic proteins and ceramide biosynthesis/metabolismduring EB and NP formation were determined. FIG. 18A shows that PAR-4expression was significantly elevated at the EB8 and NP2 stages (lanes 1and 2). By the first day of neural differentiation (D1) the PAR-4 leveldropped to less than 20% of that at NP2 (FIG. 18A, lanes 2 and 3). Thepeak time of PAR-4 expression at the NP2 stage was concurrent withcaspase 3 activation and increased PCNA (proliferating cell nuclearantigen) levels (FIG. 18A, lane 2). PCNA is a marker for mitotic cells(Chan et al.,2002 Anat. Rec., 267, 261-276), indicating that PAR-4elevation and caspase 3 activation is predominant at stages with a largenumber of proliferating stem cells. The expression of PKCζ did notchange during neuronal differentiation PKCζ activity is inhibited byceramide-enhanced binding of PAR-4 leading to an up-regulation of thecaspase 9 and 3-dependent apoptotic pathway (Diaz-Meco et al., 1998Cell, 86:777-786; Mattson, 2000 Brain Pathol., 10:300-312). Theparticipation of caspase 9 in ceramide-induced apoptosis ofdifferentiating ES cells was confirmed by the observation thatpre-incubation with the cell permeable caspase 9 inhibitor peptideLEHD-CHO suppressed apoptosis that was inducible with S18 or naturalceramide isolated from ES cells. Up-stream regulators of caspase 3, inparticular anti-apoptotic Bcl-2 and pro-apoptotic Bad, were inverselyregulated suggesting activation of caspase 3 via the mitochondrial deathpathway (FIG. 18A). However, a high degree of Bad phosphorylation (pBAD)was detectable at NP2 (FIG. 18A, lane 2), indicating the presence ofboth, apoptotic and anti-apoptotic signaling at this stage. Theexpression of Bad dropped and that of Bcl-2 increased during D1 and D4,consistent with lower levels of caspase 3 activation and apoptosis atthese differentiation stages (FIG. 18A, lanes 3 and 4).

The gene expression of PAR-4, PKCζ and serine palmitoyl transferase(SPT) that catalyzes the initial reaction of ceramide biosynthesis wereanalyzed using RT-PCR. The highest level of PAR-4 mRNA was found at theEB8 stage (FIG. 18B, lane 2). Interestingly, the level of PAR-4 mRNAdropped within 48 hours from EB8 to NP2 (FIG. 18B, lanes 2 and 3),although the NP2 stage showed the highest concentration of PAR-4 protein(FIG. 18A, lane 2). The transcription of PKCζ appeared not to bemodulated during neuronal differentiation, which is consistent with theunchanged level of protein expression. There is a more than tenfoldincrease in gene expression of SPT subunit 1-(SPT1) at the EB8 stage,whereas the transcription of the subunit 2 (SPT2) mRNA remainedunchanged at all differentiation stages from EB8 to D4 (FIG. 18B, lanes2-4). Up-regulation of SPT1 gene expression is consistent with elevatedceramide biosynthesis at EB8 and NP2 since SPT1 activates SPT2 (Hanadaet al., 1998 J. Biol. Chem., 273:33787-33794). Taken together, theresults showed that apoptosis of differentiating ES cells at the NP2stage is most likely induced by up-regulation of ceramide biosynthesisand elevation of PAR-4 expression.

Discussion

The results showed that NPs containing high levels of both ceramide andprostate apoptosis response (PAR-4) undergo apoptosis at high frequency.Several lines of evidence show that this elevation is the cause ratherthan the effect of apoptosis. Inhibition of ceramide biosynthesis withFB1 or myriocin reduces the degree of apoptosis, which can be restoredby the addition of natural ceramide or ceramide analogs. Restoration ofapoptosis is not accompanied by elevation of endogenous ceramide, rulingout a significant degree of ceramide formation by hydrolysis of ceramidederivatives in dying cells. Elevation of endogenous as well as theaddition of natural ceramide or ceramide analogs, however, restoresapoptosis only in cells with high expression of PAR-4. This is clearlyshown with NPs that were transfected with an anti-PAR-4-morpholinooligonucleotide or PAR-4-RFP cDNA before incubation with ceramideanalogs. Antisense knock-down of PAR-4 eliminates S18-inducibleapoptosis even at high concentration of the ceramide analog, whereasoverexpression results in apoptosis at low concentration of S18, butonly in PAR-4-RFP expressing cells. These experiments thus support themain hypothesis that both elevation of PARK and ceramide are necessaryto induce apoptosis.

Example 13

Asymmetric Distribution of PAR-4 and Nestin During Mitosis of NeuronalStem Cells

The apparent paradoxical elevation of both the proliferation marker PCNAand the pro-apoptotic, active caspase 3 at the EB8 and NP2 stages of EScell differentiation prompted the examination of the patterns of mitosisand apoptosis in individual cells by immunofluorescence microscopy formitosis/apoptosis markers and BrdU labeling. The predominance of mitosisor apoptosis in NPs was analyzed by immunofluorescence staining fornestin. As shown in FIG. 19, simultaneous staining by TUNEL assay andantibodies against nestin and PCNA revealed that the major portion (92%)of TUNEL positive cells were nestin-negative. Most of the TUNEL positivecells (75%) expressed PCNA as well, indicating that apoptosis followedimmediately after mitosis or during the attempt to enter the nextmitotic cycle. However, no TUNEL signal was detected if cellsco-expressed PCNA and nestin. These observations prompted theexamination of the distribution of pro-apoptotic signals inproliferating cells, in particular the expression of PAR-4 and ceramide.In double staining experiments for TUNEL and one additional marker,ceramide or PAR-4 was expressed in TUNEL-positive as well asTUNEL-negative cells (FIG. 19). The majority of the TUNEL-positive cellsexpressed PAR-4 (94%), ceramide (98%), or PCNA (75%) whereasTUNEL-negative cells showed a lower frequency for PAR-4 (27%), ceramide(34%), or PCNA (45%) expression (FIG. 19). In double stainingexperiments for two markers, PAR-4 and ceramide were independentlydistributed (31% observed vs. 24% expected frequency for the expressionof both ceramide and PAR-4, FIG. 20) within the total cell population.In triple staining experiments, however, 97% of the TUNEL-positive cellsshowed PAR-4 and ceramide expression, whereas less than 2% of theTUNEL-negative cells were stained for PAR-4 as well as ceramide. Takentogether, these results indicated that the expression of both PAR-4 andceramide was required to induce apoptosis in proliferating(PCNA-positive) stem cells or NPs. This assumption was also supported byimmunostaining for BrdU incorporation in apoptotic cells. Indifferentiating ES cells at the NP2 stage, apoptosis was observed in 70%of cells within 5 hours after BrdU labeling. Hence, in differentiatingES cells at the NP stage, apoptosis (activation of caspase 3) rapidlyfollowed mitosis BrdU incorporation).

The results from ceramide and PAR-4 double immunostaining experimentssuggested that either ceramide or PAR-4 were sequestered to only onedaughter cell during cell division. Mitotic cells in anaphase (mitosisstage VII) and telophase (mitosis stage VIII) were identified by thepolarized orientation of nuclei upon fixation and staining with Hoechstdye, a useful marker for the identification of different mitotic stages(Leblond & El-Alfy, 1998 Anal. Rec., 252:426-443). Ten out of 5000 cellswere found to be in mitosis stage VII or VIII. Ceramide was homogenouslydistributed to the two daughter cells, whereas PAR-4 was sequestered toonly one daughter cell. The daughter cells with elevated PAR-4 appearedapoptotic, as PAR-4 co-localized with ceramide in membrane blebs thatare typical for apoptosis. Ceramide-induced apoptosis in PAR-4 positivecells was executed by the caspase 9-to-caspase 3 cascade as shown byco-immunostaining of elevated PAR-4 and cleaved caspase 3 in TUNELpositive cells.

The lack of nestin expression in PAR-4 positive cells suggested thatnestin and PAR-4 were sequestered asymmetrically to the daughters ofdividing NPs. Immunostaining showed that nestin and PAR-4 were indeedasymmetrically distributed in mitosis stage VIII cells. ThePAR-4-positive, but nestin-negative cells underwent apoptosis, whereasthe nestin-positive cells were non-apoptotic and expressed either noneor significantly less PAR-4 than nestin-negative cells. The absence ofnestin-staining in apoptotic cells is consistent with the results shownin FIGS. 17, 19, and 20. These data are also consistent with the lownumber of cells (6%) that co-stained for PAR-4 and nestin (FIG. 20). Incases where residual PAR-4 was present in nestin-positive daughter cellsthe PAR-4 and nestin were clearly separated. Taken together, theseobservations suggested that PAR-4 is partitioned specifically intonestin-negative daughter cells.

Discussion

In cultures of NPs derived from ES cells it was determined that theexpression of PAR-4 and nestin is largely segregated to two separatepopulations of progenitors. Nestin(+)/PAR-4(−) cells are far less proneto apoptosis induced by high levels of intracellular ceramide, whilecells undergoing apoptosis are almost always nestin(−)/PAR-4(+). Theexpression of high levels of PARK in an apoptotic subpopulation isconsistent with previous studies demonstrating that overexpression ofPAR-4 reduces WT1- or NF-κB-mediated Bcl-2 expression, thus suppressingthe cells' self defense mechanism against ceramide-induced apoptosis(Camandola and Mattson, 2000 J. Neurosci. Res., 61:134-139; Cheema etal., 2003 J. Biol. Chem., in press). The critical role of NF-κB for cellsurvival has been shown by the observation that central neuron survivalrelies on the constitutive activity of NF-κB (Bhakar et al., 2002 J.Neurosci., 22:8466-8475). It has also been shown that elevation of PAR-4underlies neuronal cell death in several neurodegenerative diseases (Guoet al., 1998 Nature Med., 4:957-962; Xie et al., 2001 Brain Res.,915:1-10). Surprisingly, in cells that express both PAR-4 and nestin,the two proteins are strictly sequestered to different parts of thecell. This sequestration occurs already during mitotic cell division ofthe parental cells, resulting in one daughter cell that is predominantlynestin-positive, while the other one contains mainly PAR-4, but no oronly low amounts of nestin. This reveals a novel localization of apro-apoptotic signal to one of the daughter cells resulting from stem orNP cell division. The asymmetric distribution of PAR-4 in thenestin-negative daughter cells may be a mechanism to regulate the numberof differentiating cells produced from mitotically activeprogenitor/stem cells. At present, it has not been determined whetherthe asymmetric distribution results from asymmetric inheritance ofnestin and PAR-4 that have already expressed before cell division, orwhether it arises from a distinct gene expression in each daughter cellduring cell division, therefore both remain possibilities.

Example 14

A Model for Asymmetric Cell Division, Apoptosis and Differentiation

Based on these observations, a model for asymmetric cell division,apoptosis, and differentiation of neuronal stem cells is suggested,which is shown in FIG. 21. Differentiating stem cells up-regulate theexpression of nestin, ceramide and PAR-4 prior to or during celldivision. Ceramide and PAR-4 elevation at these stages is most likelycaused by up-regulation of gene expression for PAR-4 and serinepalmitoyltransferase activating subunit 1 (SPT1) in EB8. This indicatesregulation of de novo ceramide biosynthesis and PKCζ activity by therespective regulatory proteins, but not by basal enzyme activities.During mitosis, ceramide is evenly sequestered to the two daughtercells, whereas PAR-4 and nestin are asymmetrically distributed. Thenestin(−)/PAR-4(+) daughter cell undergoes ceramide-induced apoptosis,whereas the nestin(+)/PAR-4(−) daughter cell may again divide or furtherdifferentiate into a neuronal or glial precursor cell. At this point,rapid passage of the mitotic cycle is desirable in order to sequesterPAR-4 to one daughter cell and to avoid abortive mitosis beforeaccomplishment of cell division (Liu and Greene, 2001 Cell Tissue Res.,305:217-228).

There is growing evidence that elevation of ceramide is a majorregulatory factor for induction of apoptosis during in vitrodifferentiation of neuronal cells (Herget et al., 2000 J. Biol. Chem.,275:30344-30354; Bieberich et al., 2001 J. Biol. Chem., 276:44396-44404;Toman et al., 2002 J. Neurosci. Res., 68:323-330). During ongoingdifferentiation, ceramide may be converted into glycosphingolipidsand/or sphingomyelin, thereby protecting the neuroprogenitor cells fromfurther ceramide accumulation. Most recently, it has been found that thebiosynthesis of complex gangliosides from simple glycosphingolipidsactivates the IGF-1-to-MAPK cell survival pathway (Bieberich et al.,2001 J. Biol. Chem., 276:44396-44404). Further, it was suggested thatthis biosynthesis is concurrent with migration of NPs from theventricular to the intermediate zone of embryonic mouse brain. It isthus very likely that sphingolipid-dependent apoptosis of neuronal stemcells in the ventricular zone and cell survival in the intermediate zoneis regulated by a two-step mechanism. Ceramide induced apoptosis ofsubventricular stem cells relies on the simultaneous expression ofceramide and PAR-4 and its asymmetric distribution during mitotic celldivision. Only the PAR-4(+) daughter cells will undergo ceramide-inducedapoptosis and make room for nestin(+)/PAR-4(−) NPs. In the second step,ceramide is converted to ceramide-1-phosphate, glycosphingolipids and/orsphingomyelin, thus protecting progenitor cells from further ceramideaccumulation. The simultaneous elevation of ceramide and PAR-4, andsubsequent apoptosis in nestin(−) cells may thus be a mechanism toselect for nestin(+) neuronal progenitors or a mechanism to eliminateyoung post-mitotic neurons via expression of high levels of PAR-4 andceramide. In future studies, endogenous and exogenous signals thatorchestrate sphingolipid biosynthesis and the expression/asymmetricdistribution of PAR-4 and nestin during neural differentiation of stemcells in vitro and embryonic mouse brain in vivo will be determined.

1. A method of producing a human neural cell comprising, a) providing apluripotent human cell; and b) culturing the pluripotent human cell witha composition comprising a ceramide compound selected from the groupconsisting of N-(2-hydroxy-1-(hydroxymethyl)ethyl)-palmitoylamide(“S16”), N-(2-hydroxy-1-(hydroxymethyl)ethyl)-oleoylamide (“S18”),N,N-bis(2-hydroxyethyl)palmitoylamide (“B16”),N,N-bis(2-hydroxyethyl)oleoylamide (“B18”)N-tris(hydroxymethyl)methyl-palmitoylamide (“T16”),N-tris(hydroxymethyl)methyl-oleoylamide (“T18”), N-acetyl sphingosine(“C2-ceramide”), and N-hexanoylsphingosine (“C6-ceramide”) to producethe human neural cell.
 2. The method of claim 1, wherein the pluripotenthuman cell is a differentiating pluripotent human cell.
 3. The method ofclaim 1, comprising the intermediate step of forming an embryoid bodycomprising the pluripotent human cell prior to culturing a cell from theembryoid body with the ceramide compound.
 4. The method of claim 3,wherein the embryoid body is formed by culturing the pluripotent humancell with an essentially serum free medium.
 5. The method of claim 4,wherein the essentially serum free medium is a MEDII conditioned medium.6. The method of claim 5, comprising the additional steps of, a)dispersing the embryoid body to an essentially single cell suspension;b) culturing the essentially single cell suspension comprising thepluripotent human cell in an adherent culture with a compositioncomprising the ceramide compound.
 7. The method of claim 6, wherein thecomposition comprising the ceramide compound further comprises a MEDIIconditioned medium.
 8. The method of claim 5 wherein the MEDIIconditioned medium is a Hep G2 conditioned medium.
 9. The method ofclaim 7, wherein the composition comprising the ceramide compound isessentially serum free.
 10. A method of producing a human neural cellcomprising, a) providing a pluripotent human cell; and b) culturing thepluripotent human cell with a composition comprising a ceramide compoundof the structure


11. A method of producing a human neural cell comprising, a) providing apluripotent human cell; and b) culturing the pluripotent human cell witha composition comprising a ceramide compound of the structure


12. The method of claim 1, wherein the concentration of the ceramidecompound is from approximately 0.1 μM to approximately 1000 μM.
 13. Themethod of claim 1, wherein the concentration of the ceramide compound isfrom approximately 1 μM to approximately 100 μM.
 14. The method of claim1, wherein the concentration of the ceramide compound is fromapproximately 5 μM to approximately 50 μM.
 15. The method of claim 1,wherein the concentration of the ceramide compound is approximately 10μM.
 16. The method of claim 1, wherein the duration of culturing thehuman pluripotent cell with the ceramide compound is from approximately6 hours to 10 days.
 17. The method of claim 1, wherein the pluripotenthuman cell is selected from the group consisting of a human embryonicstem cell, a human inner cell mass (ICM)/epiblast cell, a humanprimitive ectoderm cell, and a human primordial germ cell.
 18. Themethod of claim 1, wherein the pluripotent human cell is a humanembryonic stem cell.
 19. The method of claim 1, wherein the humanpluripotent cell is a multipotent cell.
 20. The method of claim 19,wherein the multipotent cell is a neural precursor cell.